Expert consensus statement for basic research of animal exercise intervention studies in chronic disease prevention and treatment: A joint position paper of the Exercise Science branch of the Biophysical Society of China and the Metabolism and Genetics branch of the Genetics Society of China

Highlights

• •Physical activity has many beneficial effects for human health and is among the most cost-effective ways to prevent and treat chronic diseases.
• •Animal exercise intervention studies provide valuable scientific evidence and support for the substantial effects of exercise training on a variety of chronic disease models.
• •The consensus statement provides expert opinions and recommendations for the design and applications of appropriate animal exercise intervention studies and models in the fundamental research of prevention and treatment of chronic diseases.
• •It underlies the different animal exercise training models that are utilized for each disease and provides a list of evaluation metrics of the effects of exercise, followed by outlined recommendations for the exercise study design and evaluations for the mentioned chronic diseases.
• •The sex, age, and genetic background of animals and comparisons of different exercise models should be considered in the studies. Bridging insights from animal exercise research to human interventions offers a powerful strategy for combating chronic diseases.

Abstract

Chronic diseases, broadly defined as long-duration conditions that require sustained medical care and/or limit activities of daily living, are a major problem that threatens human health and imposes large social and economic burdens. Physical activity has many beneficial effects for human health and is among the most cost-effective ways to prevent and treat chronic diseases. Animal exercise intervention studies are widely used and provide valuable scientific evidence about the cellular and molecular mechanisms underlying the effects of exercise training in a variety of chronic disease models. This consensus statement will provide expert opinions and recommendations for the appropriate design and application of animal exercise intervention studies and models in fundamental investigations of prevention and treatment of chronic diseases, especially focusing on cardiovascular and cerebrovascular diseases (coronary artery disease and stroke), metabolic diseases (obesity and type 2 diabetes mellitus), chronic respiratory diseases (chronic obstructive pulmonary disease), and neurological diseases (Alzheimer’s disease). This statement highlights various exercise models (as determined by frequency, intensity, time, and type of exercise intervention) utilized for each disease. Additionally, it includes a list of functional, structural, biochemical, and disease-specific evaluation metrics of exercise effects, followed by outlined recommendations for the exercise study design and evaluations for the mentioned chronic diseases. This consensus aimed to offer practical recommendations for better design and conduct of fundamental research in animal exercise intervention studies to improve our understanding of the effects of exercise on chronic diseases, and to further develop physical exercise or exercise-mimetic interventions for disease prevention and treatment.

Graphical abstract

The consensus statement underlies the different exercise training models (i.e., frequency, intensity, time, and type of exercise intervention) that are utilized for the chronic diseases indicated here, and provides a list of functional, structural, biochemical, and disease-specific evaluation metrics of exercise’s effects, followed by outlined recommendations of the exercise study design and evaluations in the mentioned chronic diseases.

1. Introduction

1.1. Background of chronic diseases and physical activity

Chronic diseases are broadly defined as long-duration conditions that require sustained medical care and/or limit activities of daily living, which represent the leading cause of death worldwide. With the increasing age of populations around the world and the change in lifestyle, the prevalence of non-communicable chronic diseases continues to rise worldwide. Because of their high incidence and long duration, chronic diseases have become a major public health challenge, imposing large social and economic burdens.¹ The duration for chronic diseases is ambiguous, depending on the specific type of disease. According to different literatures, the course of chronic disease lasts for a minimum of 3 months or 1 year or longer.² But it is difficult to judge a specific endpoint in time. Major chronic diseases include cardiovascular and cerebrovascular diseases, chronic respiratory diseases, cancer, obesity, diabetes, as well as endocrine, renal, skeletal, and neurological diseases, which collectively account for over 80% of deaths in China. According to the “Healthy China 2030” Action Plan, the Chinese government has launched China’s Medium- and Long-term Plan for Prevention and Treatment of Chronic Diseases (2017–2025).³ The plan highlights health education and a healthy lifestyle as essential strategies to prevent chronic diseases. Meanwhile, it underlines the importance of early detection, standardized diagnosis, effective and timely treatment, and the whole process of health management of chronic diseases. Hence, the prevention and treatment of non-communicable chronic diseases is a major public health issue that is crucial to promoting people’s lives.

Physical inactivity is a significant risk factors for chronic diseases. According to the most recent global estimates, only 27.5% of adults meet the recommendations for aerobic exercise, which calls for urgent action to promote physical activity.⁴ The World Health Organization (WHO) launched a “Global Action Plan on Physical Activity 2018–2030”. In China, the government issued a “National Fitness Plan for 2021–2025” to promote physical activity and meet people’s health needs. Physical activity exerts many beneficial effects on an individual’s health and is among the most cost-effective ways to prevent and treat non-communicable chronic diseases.⁵ Evidence reviewed for obesity, hypertension, cancer, and type 2 diabetes mellitus (T2DM) showed beneficial effects of physical activity in decreasing the blood pressure in hypertension patients, reducing the cardiovascular risk factors and disease mortality in diabetic patients,⁶ and reducing the risk of mortality in breast cancer and colorectal cancer patients.⁷ Despite the widely known beneficial effects of physical activity, the cellular and molecular mechanisms of physical activity (or exercise training which is defined as planned and programmed physical activity) in the prevention and treatment of chronic diseases require further exploration.

1.2. Current status and challenges in animal exercise intervention studies

Animal exercise intervention studies provide valuable scientific evidence and support for the substantial effects of exercise training in a variety of chronic disease models.⁸ Basic research using animal exercise models has revealed that a systemic response to exercise can induce the release of signaling molecules, referred to as exerkines, from different tissues, such as skeletal muscle, adipose tissue, the cardiovascular system, and liver.⁹ These signaling molecules may further function in other organs and tissues through autocrine, paracrine, and endocrine pathways to regulate different diseases, like metabolic and cardiovascular diseases, musculoskeletal diseases, neurological diseases, and cancers.⁹

Commonly used animal exercise models using rodents include aerobic training (treadmill running, voluntary wheel running, and swimming exercise) and resistance training (particularly ladder climbing).¹⁰ While resistance training protocols are less common in rodent studies, they represent an important intervention modality, especially considering their clinical relevance in chronic disease management. Models of exercise can be characterized according to a group of parameters, such as exercise frequency, intensity, time, and type. Other subtle factors, such as the slope of treadmill running and the exercise environment (e.g., environment temperature, light–dark cycle, water temperature, and depth for swimming exercise) may also influence the effects of exercise training.¹¹ Since rodents are nocturnal, it is worth considering whether the exercise training will be performed during our night (their active period), which can be burdensome for investigative staff. On the other hand, exercising the rodents during our day will disrupt the normal circadian rhythm of these animals. Additionally, the sex, age, and genetic background of rodents can also influence their exercise behavior, which needs to be considered in experimental design.

Considering the clinical relevance of learning from the fundamental animal exercise intervention studies, the timing of exercise intervention in animal disease models and the correspondence between the animal exercise models and the patient exercise program are equally important issues.⁹ Hence, it is important to consider these issues when designing and performing high-quality animal exercise intervention studies to better understand the effect of exercise training in chronic diseases.

1.2.1. Treadmill running

Treadmill running is commonly used as a forced and scheduled running exercise in different species, including mice, rats, and even large animals such as pigs and horses.¹⁰ However, the most common choice for such exercise intervention studies has been rodents. In fundamental studies of the effects of exercise on chronic diseases, the choice between rats and mice might be dependent on the required disease model, the genetic background of the rodents, and the functional and structural measurements during and after exercise among other considerations. Before the formal start of treadmill running, rodents usually need a familiarization period, which can start from a low speed and short duration and progressively increase until a final protocol is achieved (e.g., 5 m/min and 10 min/day on the first day, followed by an increase of 2 m/min and 10 min/day each day until a speed of 10–20 m/min and 60 min/day for mice¹²). To ensure sustained running exercise in rodents, mild electrical stimulation (in approved jurisdictions), gently tapping their tail or hindquarters with a brush, placing a piece of gauze at the end of the treadmill, or blowing puffs of air may be effective.

The maximal oxygen uptake (VO_2max) is the maximum rate (volume per minute) at which one’s body can take up, transport and use oxygen during intense and incremental exercise. It can be measured using a metabolic chamber in animal experiments.^13,14 In terms of treadmill running, continuous moderate-intensity running (50%–70% VO_2max) and high-intensity interval running (85%–90% VO_2max) are commonly used exercise modes. However, in real world studies, there are no strict or uniform standards about the corresponding running speed to VO_2max. In general, we define intensity of treadmill running as follows: (a) low intensity (<12 m/min for mice, <20 m/min for rats, at 0%–5% slope); (b) moderate intensity (12–20 m/min for mice, 20–25 m/min for rats, at 0%–10% slope); (c) high intensity (>20 m/min for mice, >25 m/min for rats, at ≥0% slope).⁸ Rats and mice can run about 30–120 min, once or twice a day, for 5–7 days/week. The treadmill running program can last at a fixed speed and duration¹⁵ or consist of several sessions with a progressive intensity, duration, and slope of the treadmill.¹⁶ The total duration can vary from weeks to months (<6 weeks considered as short term, ≥6 weeks as long term).^17,18 One good example of the high-intensity interval training (HIIT) program in rats consists of 10 high-intensity intervals (each lasting 4 min), with 2-min active resting periods (6–8 m/min) for a total duration of 6 weeks.¹⁹ The high-intensity interval running speed is progressively increased from 12 m/min to 18 m/min within the 6-week exercise program.¹⁹ Interestingly, serum from the healthy human individuals after a single HIIT exerted the beneficial effects in amyloid-β-treated cells by promoting cell viability and reducing atrophy.¹⁹ The running speed, slope, frequency, and duration, or the time and repetitions of interval training, if applicable, are all essential parameters for treadmill running.

Endurance performance or exercise capacity can be measured after a treadmill running program as an important measurement of the effect of exercise alone or with other interventions in disease models. The endurance capacity is evaluated by the maximal run time, run speed, and total run distance to fatigue in rats or mice undergoing a treadmill exercise test. The exercise protocols vary in different studies, according to the animal, disease model, and treadmill running protocol.^14,15 The main principle is that, after familiarization to the treadmill for 5–10 min, the animal can begin to run at its accustomed speed in the daily treadmill running protocol. The speed then increases progressively every 2–5 min until the animal is exhausted. Exhaustion is evident in the behaviors when the animal can no longer keep pace with the treadmill even after several attempts to stimulate running; it can be further confirmed when a rat cannot turn over immediately when placed on its back or when a mouse fails to keep running on the treadmill after three consecutive attempts. In addition to stabilizing conditions in the running environment (e.g., room temperature (20°C–25°C) and day cycle (the rodents prefer to run during their dark cycle)), endurance exercise capacity should be performed at a fixed time of day to avoid the influences of circadian rhythm and metabolic changes (liver and skeletal muscle glycogen content) on the results.^20,21

1.2.2. Swimming exercise

Swimming is a forced systemic aerobic exercise suitable for rats and mice. Compared to treadmill running and voluntary wheel running, the experimental equipment required for swimming is very simple. However, the size of swimming equipment and environmental elements, such as water temperature and water depth, are key factors that can affect the results of animal swim training. For rat swimming, a round bucket with a diameter of 65 cm ideally allows only 2 rats to swim at the same time, ensuring a water surface area of at least 1000–1500 cm² for each rat.^22,23 For mice, swimming around a bucket with a diameter of 65 cm can accommodate 8–10 mice swimming at the same time. The recommended water temperature is between 30°C and 32°C and recommended water depth is ≥50 cm for rats and at least 10–20 cm for mice to prevent them from floating on the surface of the water.^10,11 It is noteworthy that rats will produce an evident amount of turbulence, which traps air bubbles within their fur.²⁴ The accumulation of air bubbles can cause rats to fall asleep as they float instead of swimming in the water. Thus, to establish an effective swimming exercise, it is recommended to tie a balance weight of 2%–5% of their body weight (BW) to the chest or tail (5 cm from the tail end) of the rat (approved jurisdictions), which can effectively counteract the increased buoyancy produced by the air bubbles.²⁵

Animals should be familiarized to the swimming environment for 5 min before a swimming program begins. Then animals undergo swimming exercise training, which commonly includes familiarization with swimming at the beginning. For example, rats can begin to swim for 10 min at a time twice a day, which increases 10 min per day until reaching 60 min at a time twice a day, at which point they will continue to swim at this time and frequency. An overload can be added to the rats from the third day of swimming by introducing an additional 3% BW load. The total duration of swimming exercise can be several weeks, including the adaptation program.²⁵ As for mice, they can begin to swim for 10 min at a time twice a day, progressively increasing by 10 min per day until reaching 60–90 min at a time twice a day. Mice will continue to swim with this program for a few weeks according to different experimental designs.26, 27, 28 A minimum of 3–4 weeks (once or twice a day) of such a swimming program is considered chronic exercise training in rodents, which could correspond to chronic aerobic exercise in humans.

The experimenters should strictly maintain the water temperature and observe the swimming performance of the animals. Animals climbing on the bucket wall, thermometer, or other animal’s back need to be stopped in a timely fashion. The experimenters can use a bottle brush to gently keep animals swimming. Consider excluding animals that frequently show distress during the familiarization/training period, as this can affect swimming performance and increase variability of the exercise-induced response. After each swim, animal hair should be individually dried with a towel or wiped with a towel and then dried with a warm air blower (use the low temperature gear and keep at least 20–30 cm away from animals).

1.2.3. Voluntary wheel running

Distinct from forced treadmill running or swimming, voluntary wheel running represents an autonomous form of exercise that allows animals to choose when and how much they want to exercise. This autonomy can lead to more naturalistic behaviors and potentially different physiological adaptations. Prior to data collection, a 1–2-day adaptation period is essential for animals to acclimate to the running wheel and experimental conditions. To achieve good running results, the size of the stainless-steel running wheel is an important consideration. Running on a voluntary wheel that is 11 cm in diameter and 5 cm in wheel width for 8 weeks can increase cardiomyogenesis in mice.²⁹ Wheels that are ∼35.5 cm in diameter and ∼10.0 cm in wheel width are suitable for rat comfort.³⁰ Animals should be inspected for broken toenails or hind-paw injuries that may occur over time and impede their ability to exercise.

It is recognized that rodents are social animals, so the experimental rodents have better welfare when they are housed in pairs or groups.³¹ It is noteworthy that most rodent exercise studies using voluntary wheel running housed exercised animals individually.32, 33, 34 However, some strategies are useful to reduce the influence of an isolated housing environment. A pre-adaptation to the voluntary wheel running environment is essential for the animals before they formally begin the exercise program. Moreover, environmental enrichment materials such as enrichment toys can be put in the cage.³⁴ In most cases, there is no significant evidence showing that the individual housing of animals under voluntary wheel running will influence the effect of exercise on chronic diseases, with the important exception of investigations into brain development and behavior or psychiatric disorders.^35,36

The running activity is controlled by recording a group of parameters, such as instantaneous speed and maximum speed, run time, and run distance. Although these parameters are not commonly provided by the authors in their published work, some studies have reported that rats performing voluntary wheel running display different running activity: (a) low activity (2–5 km/day); (b) moderate activity (>5–11 km/day); (c) high activity (>11 km/day).³⁷ Adult C57BL/6 mice that run at a speed <3.1 km/day can be excluded from the study.³⁸ Total wheel running durations varied in different studies regarding the observations of interest. It has been reported that C57BL/6J and BALBc/J mice can run vigorously from the beginning of voluntary wheel running (∼4–5 km/day). However, Wistar or Sprague-Dawley (SD) rats run ∼1 km/day on their first days of running.^39,40 After 4 weeks of voluntary wheel running, mice and rats run at a relatively stable level.^39,40 The respiratory monitoring, food intake, and telemetry physiological indicators can also be measured by adding other modules to the system. The major drawback of voluntary running is that the investigator cannot precisely control the exercise “dose”. If a particular line or genetic model chooses to exercise less (or more) than controls, it can be hard to disentangle this confounder from any differences in observed effects. Additionally, sex differences also influence the exercise behavior patterns of mice with voluntary wheel running.⁴¹ Female mice spend more time engaged in voluntary wheel running and have a higher average running speed at the first 2 weeks than male mice. By the third week, the average running speed is similar between male and female mice. However, female mice still spend more time running than males, thus female mice keep running greater distances than male mice on the voluntary running wheel.⁴¹

1.3. Objectives of the consensus statement

The objective of the consensus statement was to provide expert opinions and recommendations for the design and applications of appropriate animal exercise intervention studies and models in the fundamental research of prevention and treatment of non-communicable chronic diseases. In this document, we will specifically focus on cardiovascular and cerebrovascular diseases (coronary artery disease (CAD), stroke), metabolic diseases (obesity, T2DM), chronic respiratory diseases (chronic obstructive pulmonary disease), and neurological diseases (Alzheimer’s disease (AD)).

The consensus statement underlies the different exercise training models (i.e., frequency, intensity, time, and type of exercise intervention) that are utilized for each disease and provides a list of functional, structural, biochemical, and disease-specific evaluation metrics of the effects of exercise, followed by outlined recommendations for the exercise study design and evaluations for the mentioned chronic diseases (Fig. 1).

Fig. 1.

Animal exercise intervention studies for chronic diseases.

2. Animal exercise intervention studies for cerebrovascular and cardiovascular diseases

2.1. Stroke

2.1.1. Introduction to stroke

Stroke is a medical emergency that occurs when the brain’s blood supply is suddenly interrupted or when a blood vessel in the brain ruptures and bleeds, often resulting in neurological dysfunction. Since the early 20th century, stroke mortality has ranked among the top 5 causes of death in the USA. From 2011 to 2021, the age-adjusted stroke death rate in the USA increased by 8.4% (from 37.9 per 100,000 to 41.1 per 100,000), and the actual number of stroke deaths increased by 26.3% (from 128,932 to 162,890).⁴² In China, the incidence and mortality of stroke are significantly higher than the global average. The overall incidence rate of stroke among individuals aged 40 and above is 500 cases per 100,000 person-years in China, which is considerably higher than global estimates of 158 per 100,000 person-years.^43,44 Furthermore, stroke has become the leading cause of premature death in China. A report in 2019 indicated that stroke is the leading cause of disability-adjusted life years in China, surpassing other conditions like heart diseases and cancers of the respiratory or digestive systems.⁴⁵

The pathogenesis of stroke is complex, involving multiple pathophysiological and molecular pathways such as energy metabolism disorders, excitotoxicity, oxidative stress, inflammatory responses, autophagy, apoptosis, and necroptosis.⁴⁶ Initially, during a stroke, the interruption of blood flow to the brain results in a sudden decrease of oxygen and glucose supply to the brain. This will cause metabolic disruption and impaired adenosine triphosphate (ATP) synthesis and will subsequently affect neuronal functions and activities.⁴⁷ Meanwhile, excitatory neurotransmitters like glutamate are substantially released, leading to neuronal overexcitation, or excitotoxicity, which damages the neurons.^48,49 Moreover, mitochondrial dysfunction and uncoupling of oxidative phosphorylation increase reactive oxygen species (ROS) generation, which can further lead to lipid peroxidation and high cell membrane permeability as well as DNA breaks and protein oxidation.⁵⁰ Upon a stroke, the release of damage-associated molecular patterns also triggers local inflammation in the affected brain region, amplifying secondary injury by aggravating blood–brain barrier disruption, microvascular failure, cerebral edema, and oxidative stress.⁵¹ Ultimately, these damage signals can activate apoptotic pathways, which lead to programmed neuronal death and further worsen brain tissue damage.⁵² These mechanisms intertwine to form a complex pathological network during the occurrence and development of stroke. Interventions that target any of these aspects may hold promise for improving the outcomes of stroke.

2.1.2. Epidemiological studies of exercise intervention in stroke

Exercise intervention has become a vital component in the rehabilitation of stroke patients that may provide benefits for both physical and cognitive recovery. A substantial body of evidence also supports physical activity as a critical element in primary stroke prevention strategies. Notably, high-intensity physical activity is associated with a 27% reduction in the risk of stroke or death, and moderate-intensity activity levels also correlate with significant risk reductions.⁵³ Individuals engaging in moderate-intensity physical activities, especially those who accumulate 5–10 metabolic equivalent (MET) hours of activity per day, demonstrate lower risks for total stroke, hemorrhagic stroke, and ischemic stroke.53, 54, 55 Furthermore, exercise with combined aerobic and strength training positively impacts cognitive and motor functions post-stroke and even during the chronic phase after stroke.⁵⁶ Exercise or physical activity can also reduce the risk of stroke recurrence.⁵⁶ For instance, a meta-analysis has shown that various upper limb exercise interventions effectively restore motor function and enhance the quality of life in individuals with acute or subacute stroke.⁵⁷ These findings collectively underscore the essential role of exercise intervention for both stroke prevention and recovery, highlighting the need for its integration into clinical management and public-health strategy.

2.1.3. Basic research on the effect of exercise in stroke

Animal studies indicate that exercise intervention, whether conducted before or after stroke onset, can effectively reduce neurological damage.^58,59 Brain-derived neurotrophic factor (BDNF) plays a crucial role in neuronal survival, differentiation, and synaptic transmission. Exercise can increase the expression of BDNF, vascular endothelial growth factor (VEGF), growth-associated protein 43 (GAP43), and myokine irisin, whereas it reduces plasma fibrinogen expression, thereby inhibiting stroke-induced neuronal death and promoting neurogenesis and synaptic plasticity.58, 59, 60, 61, 62

Moreover, exercise can reduce inflammatory responses and lower levels of inflammatory factors such as nucleotide-binding oligomerization domain (NOD)-, leucine-rich repeat (LRR)- and pyrin domain-containing protein 3 (NLRP3), tumor necrosis factor-α (TNF-α), and interleukin (IL)-1β, thus mitigating inflammation-mediated neuronal damage.^63,64 Exercise also exhibits antioxidant effects by increasing the activity of antioxidant enzymes like superoxide dismutase (SOD) post-stroke.^65,66 Meanwhile, exercise improves vascular function and promotes angiogenesis, which enhances blood flow to the injured brain and provides essential oxygen and nutrients for neural repair as well.^58,67,68

In summary, exercise can mitigate stroke pathogenesis through various mechanisms, including promoting neural plasticity, reducing inflammatory responses, exerting antioxidant effects, and enhancing vascular function. These research findings provide important theoretical support for the benefits of exercise in stroke.

2.1.4. Animal exercise intervention studies and models for stroke

Middle cerebral artery occlusion (MCAO) and photothrombotic cerebral infarction are commonly used animal models for studying stroke in mice or rats. When exploring the effects of exercise on stroke, aerobic exercise is widely recognized as an effective strategy for improving motor function recovery after a stroke. Various exercise types, such as treadmill running, swimming, and voluntary wheel running, have been utilized in rat and mouse models to assess their protective functions against stroke-related damage.62, 63, 64, 65^,69 In earlier stroke research, rotarod running was employed as a mode of exercise, showing neuroprotective effects post-stroke.^70,71 Compared to treadmill running, rotarod running requires more precise limb coordination, making it a more complex exercise type. Additionally, this exercise mode typically does not induce changes in cardiac structure or function, nor does it affect the oxidative capacity of cardiac or skeletal muscle tissues.⁷² Given the limited research on this exercise mode, rotarod exercise will not be involved here.

Aerobic exercise is predominantly implemented through treadmill interventions in stroke research, typically involving low-, moderate-, and high-intensity exercise in adult mice or rats. These exercises last at least 2 weeks, with speeds ranging from 10 m/min to 30 m/min and running sessions from 30 min/day to 90 min/day.^18,61, 62, 63^,65 In addition to treadmill running, swimming has emerged as another aerobic exercise mode that is beneficial for stroke. Studies typically involve adult rats with varying swimming sessions lasting between 5 and 20 min/day over a period of from 3 days to 4 weeks. One study investigated the effect of swimming exercise using 3 protocols (Group 1: 5 min/day; Group 2: 10 min/day; and Group 3: 10 min twice daily) and assessed neurological function and acute neuroprotective gene expressions (semaphorin 3A (Sema3A) and neuropilin-1 (NRP-1)) at 3 days, 7 days, and 14 days after stroke.⁷³ Another study examined long-term rehabilitation outcomes using swimming time-based protocols (short: 5 min/day; moderate: 10 min/day; long: 20 min/day) over 4 weeks, focusing on neuroplasticity markers (BDNF and VEGF).⁶⁹ Voluntary exercise using wheel running represents a third exercise mode applied in stroke research. This exercise program typically spans from 2 to 4 weeks, allowing animals to engage in physical activity at their discretion.^64,74 The following sections will introduce the key points related to treadmill running, swimming, and voluntary wheel running in stroke research (Supplementary Table 1).

2.1.4.1. Treadmill running

Treadmill running has become the primary type for research on the effects of exercise on stroke. Male C57BL/6J mice aged 4–12 weeks and SD or Wistar rats weighing 230–350 g and aged 8–12 weeks are typically used for these experiments. It is important to note that pre-training before any exercise protocols needs to be implemented. The pre-training generally lasts 3 days and is conducted at lower speed (4–15 m/min or at the animal’s preferred pace) with shorter exercise durations (10–30 min/day) to acclimate them to the exercise equipment.^62,63,65,75 For instance, after 3 days of adaptation to treadmill training before surgery, rats were exercised on a four-lane treadmill at a speed of 12 m/min for 30 min/day, 5 days/week, starting 3 days post MCAO-reperfusion.⁷⁶ This pre-training is recommended to familiarize the stroke animals with the treadmill apparatus, ensuring that they can engage in subsequent experimental protocols without undue stress or discomfort. Additionally, exercise intensity can be assessed according to Bedford’s theory,⁷⁷ which categorizes intensity as <50%, 50%–75%, or >75% VO_2max. This categorization allows for the implementation of low-, moderate-, and high-intensity treadmill running exercises to investigate their specific effects on stroke prevention or recovery.

Low-intensity: Low-intensity treadmill interventions in stroke-related studies commonly employ training protocols at speeds of 10 m/min for 30–90 min/day over 2–3 weeks in male C57BL/6J mice (aged 4–12 weeks),^62,65 or at speeds of 12–20 m/min for 30 min/day, 5–7 days/week, over 1–8 weeks in SD or Wistar rats weighing 230–350 g and aged 8–12 weeks.^{18,67,68,75,76,78,79} For example, pre-training C57BL/6J mice at 5 m/min for 30 min/day over 3 days before stroke induction, followed by low-intensity maintaining at 10 m/min for 30 min/day over 3 weeks (5 days/week), resulted in improved neurological function and decreased neuroinflammation.⁶⁵

Moderate-intensity: Moderate-intensity treadmill running in stroke-related studies typically employs training protocols with speeds of 20–24 m/min for 30 min/day over 3–4 weeks in SD male rats (aged 8–12 weeks).^18,63 For instance, pre-training animals at 10 m/min, 30 min/day for 3 days and initiating treadmill exercise intervention before MCAO at 20 m/min, 30 min/day for 3 weeks (6 days/week) led to reduced neurological deficit scores and brain injury in SD rats, along with decreased Bcl-2-associated X protein (Bax) and increased B-cell lymphoma 2 (Bcl-2) expressions.⁶³

High-intensity: Research also indicates that high-intensity exercise can enhance recovery in stroke models, with speeds of 30 m/min for 30–50 min/day over 4–8 weeks in male SD rats (aged 10 weeks) or male Wistar rats (260–300 g).^18,61 One study implemented a protocol that included pre-training at 15 m/min (slope = 0°) for 10–15 min/day over 3 days.⁶¹ Twenty-four hours after cerebral ischemia/reperfusion (I/R) injury, exercise began at a speed of 18 m/min for 20 min/day at a 0° slope during the first week, and was then gradually increased to 30 m/min for 50 min/day (5 days/week) at a 10° slope from the second to eighth week.⁶¹ This high-intensity exercise regimen significantly elevated the levels of BDNF and VEGF in the hippocampus of stroke-affected rats, reducing cell death and improving motor function as well.⁶¹

2.1.4.2. Swimming

In stroke-related studies, swimming has also been used as an exercise protocol in 220–350 g male SD rats for durations ranging from 3 days to 4 weeks.^69,73 In a study designed to determine the maximal endurable duration in a water pool, nearly all the photothrombotic cerebral infarction rats drowned when the exercise time exceeded 20 min.⁶⁹ Therefore, a long swimming session was set at 20 min for these rats, a moderate session at 10 min, and a short session at 5 min. The swimming exercises were conducted in a round water tank, with a diameter of 184 cm and a water depth of 30 cm.⁶⁹ It is important to note that these classifications are based on the exercise session (or time) rather than true exercise intensity. In cases involving more severe MCAO models, swimming sessions were typically limited to twice daily, with each session lasting 10 min, as part of the overall swimming program.⁷³

2.1.4.3. Voluntary wheel running

Voluntary exercise utilizing wheel running is frequently employed in research examining the preventive and rehabilitative effects of exercise on stroke in male C57BL/6J mice (aged 6–8 weeks) or SD rats (380–420 g).^64,74 Compared to treadmill interventions, the neuroprotective effects of voluntary wheel running typically require a duration of around 2–4 weeks to manifest significant benefits.^64,74 For example, the wheel running apparatus used for SD rats had a diameter of 34.0 cm and a width of 9.0 cm, and it was fitted with an acrylic cage (330 mm wide × 115 mm long × 125 mm high). To minimize novelty stress, all rats were habituated to the apparatus for 3 days prior to infarction surgery. The voluntary exercise commenced 2 days after surgery for the exercise group and was carried out during the dark period. The exercise duration was set at 12 h/day, 7 days/week, lasting for 14 days to analyze motor behavior or lasting for 5 days for biochemical assays. Results indicated that voluntary wheel running significantly improved motor ability in stroke-affected rats and increased the expression of GAP43 and phosphorylated GAP43 in the infarct perilesional cortex.⁷⁴

2.1.4.4. Intervention time

It is important to note that the timing of exercise interventions following a stroke is critical. Ideally, these interventions should begin within 5 days after a stroke.^{61,68,70,75,76,78} Delayed interventions, such as those started more than 5 days post-stroke, may not yield the protective effects associated with exercise.^70,80

2.1.5. Evaluation metrics in stroke research with an exercise intervention

In the prevention and treatment of stroke, exercise has shown significant potential as a non-pharmacological intervention. To assess the effect of exercise on stroke, researchers typically employ a series of behavioral tests and biomarkers for a comprehensive evaluation (Table 1). Behavioral tests, such as the novel object test, Y-maze test, rotarod test, and beam-walking test, aim to quantify the learning ability, memory, and motor coordination of the mice.^{61,62,64,65,68,74,75,78} The 2,3,5-triphenyltetrazolium chloride (TTC) staining is used to visualize brain infarction volume.62, 63, 64^,68,74,75,78 Additionally, laser speckle contrast imaging allows for real-time measurement of cerebral blood flow, providing insights into blood flow recovery post-exercise.⁶⁵ For evaluating post-stroke angiogenesis, vascular endothelial cell markers such as CD31/CD34 and factor VIII-related antigen (FVIII-R Ag) are utilized to assess the effect of exercise on vascular generation.⁶⁸ Furthermore, key inflammatory markers, including various microglial markers (ionized calcium-binding adapter molecule 1 (Iba-1) for total microglia, CD86 for M1-type microglia, arginase-1 (Arg1) for M2-type microglia), oligodendrocytes, and infiltrating neutrophil marker (MPO-1) and pro-inflammatory or anti-inflammatory cytokines (IL-6, IL-1β, IL-10, and NLRP3), reflect inflammation levels following a stroke.62, 63, 64, 65 Collectively, the integration of behavioral tests, TTC staining, cerebral blood flow measurement, angiogenesis markers, and inflammatory biochemical markers offers a comprehensive evaluation of the effects of exercise. This multifaceted approach enhances our understanding of the mechanisms through which exercise supports stroke rehabilitation.

Table 1.

Evaluation metrics in stroke research that could be examined with an exercise intervention.

Detection index	Parameters	Reference
Behavior function  Memory function  Motor function  Neurological function scale	Novel object recognition test, Y-maze test Skilled ladder rung walking test, rotarod test, beam-walking test, grip test, adhesive removal test Longa’s neurological deficit test, mNSS test	62, 63, 64, 65,68,74,75,78
Histological analysis	H&E staining for morphological examination: assessment of neuronal damage, tissue necrosis, and overall tissue architecture TTC staining for infarct size	62, 63, 64, 65,68,74,75,78
Vascular network	Cerebral blood flow determination by LSCI CD31/FVIII-R Ag (vascular endothelial cell marker)	65,68
Inflammatory response	Iba-1/CD86/Arg1 (microglia/M1-type/M2-type microglia marker) MPO-1 (oligodendrocytes and infiltrating neutrophils marker) Expression of IL-10, TNF-α, IL-6, IL-1β, IL-1β, and NLRP3, et al.	62, 63, 64, 65
Oxidative stress	Expression of iNOS, SOD1, NO2-Tyr, superoxide anion, 4-HNE and MDA DHE staining	62,63,65
Cell death	TUNEL staining FJB staining	62,63

Abbreviations: 4-HNE = 4-hydroxynonenal; Arg1 = Arginase 1; CD31 = cluster of differentiation 31; CD86 = cluster of differentiation 86; DHE = dihydroethidium; FJB = Fluoro-Jade B; FVIII-R Ag = factor VIII-related antigen; H&E = hematoxylin–eosin; Iba-1 = ionized calcium-binding adapter molecule 1; IL= interleukin; iNOS = inducible nitric oxide synthase; LSCI = laser speckle contrast imaging; MDA = malondialdehyde; mNSS test = modified neurological severity score test; MPO-1 = myeloperoxidase-1; NLRP3 = nucleotide-binding oligomerization domain (NOD)-, leucine-rich repeat (LRR)- and pyrin domain-containing protein 3; NO₂-Tyr = nitrotyrosine; SOD1 = superoxide dismutase 1; TNF-α = tumor necrosis factor-α; TTC = 2,3,5-Triphenyl tetrazolium chloride; TUNEL = terminal deoxynucleotidyl transferase 2’-deoxyuridine 5’-triphosphate (dUTP) nick end labeling.

2.1.6. Recommendations

• •Various exercise types, including treadmill, swimming, and voluntary wheel running interventions, have shown significant protective effects against stroke.62, 63, 64, 65
• •In terms of treadmill running exercise, moderate-intensity exercise promotes neuroplasticity more effectively than low-intensity or high-intensity exercise.¹⁸
• •When using swimming protocols, swimming duration should be limited to ≤20 min, as stroke rats may exceed their maximal endurable duration and risk drowning beyond this threshold.⁶⁹
• •The implementation of treadmill^{61,68,75,76,78} and voluntary wheel⁷⁴ running exercise after a stroke is crucial. Early interventions (within 5 days after a stroke) significantly enhance motor function and reduce brain infarct area.
• •In therapeutic research, treadmill running^68,75,78 and swimming⁷³ exercise interventions with a short duration (approximately 1–2 weeks) can produce neuroprotective effects by reducing cerebral ischemic infarction and promoting new blood vessel formation.
• •For preventive effects, treadmill running exercise with longer duration (at least 3 weeks) is recommended.^61,65,67 Therefore, exercise protocols should consider exercise duration to optimize the neuroprotective benefits of exercise.
• •VO_2max is a key indicator for assessing aerobic exercise intensity, yet the detection and control of VO_2max are rarely mentioned in stroke research, which warrants more attention.

2.2. Coronary artery disease (CAD)

2.2.1. Introduction to CAD

Coronary artery disease is characterized by the impairment of blood vessels supplying the heart, which is primarily caused by atherosclerosis of coronary arteries. It may cause stable angina, acute coronary syndrome (unstable angina, myocardial infarction (MI)), chronic heart failure, or even sudden cardiac death.^81,82 As one of the most common cardiovascular conditions, CAD has become the leading cause of death globally. The global prevalence of CAD was estimated as 315 million (95% uncertainty interval ranging from 273 million to 362 million) in 2022.⁸³ According to the Global Burden of Disease study, the mortality rate of coronary heart disease, also known as ischemic heart disease, was 108.7 per 100,000 members of the population in 2021.⁸⁴ The prevalence of CAD in China has consistently increased as well. It is estimated that approximately 11.39 million individuals in China suffer from CAD, which was slightly lower than the 13 million individuals affected by stroke. CAD is the second leading cause of death in China.⁸⁵

CAD is related to atherosclerosis, a pathological condition characterized by the formation of plaques in the coronary arteries that narrow the vessel lumen and reduce blood flow to the myocardium.⁸⁶ The pathogenesis of CAD involves several interrelated mechanisms that contribute to the development and progression of atherosclerosis.⁸⁶ Risk factors such as hypertension, cigarette smoking, and elevated cholesterol levels may induce damage to the endothelium and increase its permeability.⁸⁷ Lipids, especially low-density lipoprotein (LDL) cholesterol, penetrate the damaged endothelium and undergo oxidation. The oxidized LDL is detrimental, triggering inflammatory responses within the arterial wall. Macrophages migrate to the site of injury to engulf oxidized LDL, transforming into foam cells that contribute to plaque formation.^88,89 These foam cells accumulate along with other cellular debris to form fatty streaks, which finally develop into larger plaques.⁹⁰ The plaques may rupture and lead to thrombus formation, partially or completely obstructing blood flow in the coronary arteries and inducing myocardial ischemia.^91,92 Reduced blood flow typically manifests as angina pectoris, and complete obstruction results in MI (heart attack).⁹³ The major risk factors contributing to the development of atherosclerosis and CAD include obesity, diabetes, tobacco exposure, hypertension, and dyslipidemia (elevated levels of cholesterol and other lipids).⁹⁴

Treatment for CAD includes a variety of medications and medical procedures aiming to reduce myocardial ischemia and improve coronary blood flow.^92,95 Physical exercise and lifestyle changes have been found to be effective as medications.⁹⁶ Regular physical activity reduces the risk of CAD by approximately 25%.⁹⁷ Prevention of CAD involves adequate physical exercise, weight management, control of hypertension, a healthy diet, and smoking cessation. Many individuals with CAD can maintain active and fulfilling lives with appropriate intervention.⁹⁸ However, CAD remains a leading cause of morbidity and mortality globally. A thorough understanding of these processes is crucial for the development of effective management strategies for CAD.

2.2.2. Epidemiological studies of exercise intervention in CAD

Regular physical activity exerts positive effects with respect to maintaining cardiovascular health and reducing CAD risk factors and all-cause mortality.^99,100 It is recognized as an effective preventive strategy for CAD and is recommended as a therapeutic intervention for managing CAD in clinical guidelines.¹⁰¹ Moderate- and high-intensity aerobic exercise trainings provide significant benefits for patients with CAD,¹⁰² including improvement of functional capacity, health-related quality of life, blood rheology, estimated metabolic equivalents, maximal oxygen uptake, and peak anaerobic threshold. These physiological enhancements contribute to reduction of morbidity, mortality, hospitalization, and associated health care costs.^99,100,103 Regular physical activity also exhibits positive effects on lipid profiles by regulating plasma total cholesterol, triglycerides, LDL, and high-density lipoprotein (HDL). Aerobic exercise has been shown to reduce insulin resistance and body mass index (BMI).¹⁰⁴ In addition, physical activity can effectively slow the progression of rising blood pressure and improve the remission of hypertension.^6,105 Although higher levels of physical activity are associated with lower mortality rates in CAD patients, even lower doses (frequency, intensity, and duration) of physical activity are able to exert benefits.¹⁰⁶ Compared to a sedentary lifestyle, engaging in lower-intensity daily activities, like office-based physical activities, walking, and climbing stairs, can improve energy expenditure and functional capacity, decrease cardiometabolic risk, and reduce the incidence of cardiovascular events substantially.107, 108, 109 In addition to improvements of functional and biochemical parameters, exercise also decreases psychological risk factors and promotes better mental health outcomes in CAD patients.¹¹⁰ Cardiac rehabilitation with exercise training markedly reduces the prevalence of anxiety.¹¹¹ Collectively, exercise and physical activity have positive effects on the prevention and treatment of CAD.

2.2.3. Basic research on the effect of exercise in CAD

Animal exercise models have been widely used to explore the functional and molecular mechanisms of exercise-induced protective effects in CAD.¹¹ Substantial experimental studies have demonstrated that regular exercise exerts positive effects on myocardial perfusion and endothelial function.¹¹² Due to the increased demand for oxygen and energy during exercise, the cardiovascular system responds with certain adaptive changes. Exercise increases systemic blood flow and shear stress, which stimulates the endothelium to secrete vasodilators (e.g., nitric oxide (NO)) and further promotes smooth muscle cell relaxation and vasodilation.¹¹² Additionally, exercise improves coronary artery stiffness and endothelial aging, and increases the sensitivity and maximal responsiveness of resistance vessels to adenosine, thus consistently contributing to enhanced endothelium-dependent vasodilation.^113,114 Moreover, exercise upregulates the expression levels of VEGF receptors (specifically, VEGFR-1 and VEGFR-2)¹¹⁵ and skeletal muscle-derived follistatin-like-1 (FSTL1)¹¹⁶ in the heart, which facilitate endothelial cell proliferation and myocardial angiogenesis. In mice with physiological cardiac hypertrophy induced by swimming, exercise also induces cardiac lymphangiogenesis through VEGFR-3 activation.²⁶

Meanwhile, the protection of exercise against CAD is related to exercise-enhanced myocardial antioxidant capacity.¹¹⁷ Exercise intervention studies on treadmill running in rodents have proved that exercise effectively increases the expression and activity of key antioxidant enzymes, such as SOD, glutathione peroxidase, and catalase in the mitochondria of ventricular myocytes, resulting in reduced mitochondrial ROS generation and, subsequently, smaller infarct size following myocardial I/R injury.¹¹⁸

Exercise-induced physiological cardiac hypertrophy and its related molecular mechanisms provide novel strategies and potential therapeutic targets for the management of CAD.¹¹⁹ Moreover, exercise contributes to cardioprotection by promoting metabolic adaptations in the heart and modulating gut microbiota composition.²⁶ Regular exercise also maintains cardiovascular health through regulating the immune system as well as stimulating the release of myokines and metabolites.¹²⁰

2.2.4. Animal exercise intervention studies and models for CAD

Hypercholesterolemic apolipoprotein E (ApoE) knockout (KO) mice and LDL receptor (LDL-R) KO mice, which develop spontaneous and diet-accelerated atherosclerosis,121, 122, 123 and mice subjected to MI or I/R injury by left anterior descending coronary artery ligation (LAD) surgery124, 125, 126 are widely used animal models in CAD research. To investigate the effect of exercise on CAD, various exercise types, such as treadmill running, voluntary wheel running, and swimming, have been applied.^122,126, 127, 128 Male mice are more frequently utilized in experimental studies of CAD because they maintain relatively stable levels of steroid hormones, while females experience hormonal fluctuations throughout their menstrual cycle, which may increase variability in study outcomes.¹²⁹ However, it has been demonstrated that female mice exhibit greater increases in VO_2max, ventricular mass, and cardiomyocyte size compared to male mice after an intensity-controlled treadmill running program.¹³⁰

Treadmill running exercise has been shown to exert positive effects on cardiac function in CAD studies. Moderate-intensity treadmill running in mice is commonly performed at a speed of 12–16 m/min, 60–90 min/day, for 4–12 weeks.^121,122,131 Alternatively, HIIT is applied by alternating between high-intensity running exercise (80%–90% VO_2max) and interval low/moderate-intensity exercise (40%–60% VO_2max), 50–60 min per day, for 4–8 weeks.^125,132,133 These treadmill training programs exert beneficial effects to improve cardiac function and attenuate myocardial damage in animal models of CAD, including MI^125,132,133 and atherosclerosis,¹²³ and to alleviate endothelial dysfunction in a model of familial hypercholesterolemia.¹²² Swimming is another exercise model used to mimic aerobic exercise in animals. A typical swimming program is conducted 45–90 min per session, once or twice daily, for a total duration ranging from 3 to 16 weeks.^128,134 Voluntary wheel running is frequently employed in CAD studies to investigate the effects of voluntary exercise on CAD. This typically involves providing unrestricted access to a running wheel for a minimum of 4 weeks.^126,135,136 The key parameters of exercise training by treadmill running, swimming, and voluntary wheel running in animal models of CAD are summarized in Supplementary Table 2.

2.2.4.1. Treadmill running

In CAD studies, treadmill running is commonly used in the form of both long-term moderate-intensity aerobic exercise and HIIT to investigate the mechanisms underlying exercise-induced cardioprotection.¹¹ In such research, the animal models of CAD—such as MI induced by LAD,¹²⁵ atherosclerosis induced by gene deficiency (ApoE^-/- mice¹²³) or diet (i.e., high-fat diet (HFD)¹³¹), and the transgenic hypercholesterolemia model (LDL-R^-/- mice¹²²)—at 8–12 weeks of age are frequently utilized. It should be noted that a pre-training period of 3–7 days is required before the formal exercise program begins. Following this adaptation, VO_2max can be measured by an acute incremental exercise test on the treadmill. The measurement can start from a speed of 5 m/min and then increase continuously by 3.3 m/min every 3 min at a 0% gradient until the exhaustion of animal, defined as an inability to maintain the required running speed.¹²² The protocols for treadmill running in CAD animal models vary in different study designs, which can be categorized as moderate-intensity (50%–70% VO_2max) and high-intensity (85%–90% VO_2max). Each treadmill running session can vary from 30 min to 2 h, with most training protocols lasting 60–90 min per session to elicit typical adaptations associated with endurance training.¹⁰ The frequency of training may range from once or twice daily to every other day, depending on the targeted outcomes (e.g., improved exercise tolerance, exercise-induced cardiac hypertrophy).¹⁰ For instance, in studies investigating the cardioprotective effects of exercise against MI, both moderate-intensity running (60% VO_2max) and HIIT, performed for 60 min once daily over 8 weeks, can preserve cardiac function post-MI.¹²⁵

Moderate-intensity: Treadmill running is used as a common exercise mode for investigating the effects of exercise on cardiovascular changes. In studies using moderate-intensity running trainings in CAD models, exercise protocols are generally performed at a speed achieving 50%–70% VO_2max, 50–90 min/day (5–7 days/week), over a period of 4–12 weeks.^{121,122,131,137,138} For example, LDL-R^-/- mice first undergo an adaptation training at a speed of 10 m/min; the training session lasts 10 min on Day 1 and gradually increases to 30 min by Day 4. Then the treadmill running program starts with the speed and duration at 10 m/min for 30 min per day, progressively increases to 15.0–16.6 m/min (equivalent to 60%–70% VO_2max) for 60 min per day, and lasts 4 weeks in total. This exercise improves endothelial dysfunction in the aortas of mice at the early stages of atherosclerosis.¹²² Moreover, in the HFD-induced atherosclerosis rat model, 60% HFD-fed rats that perform treadmill running at 16 m/min (exercise intensity corresponding to 65%–70% VO_2max), 60 min/day, for 8 weeks are able to inhibit BW gain and the balloon-induced neointimal formation, and to reduce LDL-R expression in the liver.¹³⁷ There is no standardized requirement for the incline of the treadmill.

High-intensity: A previous study subjecting rats to treadmill running at a speed of 28 m/min after coronary stenosis demonstrated that high-intensity running exercise for 5 min/day over 12 weeks reduced cardiac dysfunction and remodeling and improved myocardial NO activity in the rats with coronary stenosis.¹³⁹ Meanwhile, running at 28 m/min for 15 min/day for 12 weeks aggravated cardiac dysfunction and remodeling in these animals.¹³⁹ Thus, instead of continuous running, high-intensity treadmill running is commonly performed by HIIT in CAD researches. The HIIT program is conducted by alternating between high-intensity running (corresponding to 80%–90% VO_2max) and interval low/moderate intensity running (corresponding to 40%–60% VO_2max), with each session lasting approximately 50–60 min per day for 4–8 weeks.^125,132,133 To improve cardiac function and exercise capacity in MI, HIIT training in rats was performed on a treadmill with a 0°–15° incline under the following protocol: 7 cycles of 3 min at 60% VO_2max and 4 min at 85%–90% VO_2max for a total duration of 49 min/day, 5 days/week, for 4 weeks.¹³² Both HIIT and moderate-intensity aerobic exercise can improve cardiac function and structure, attenuate oxidative stress, and improve glucolipid metabolism and exercise tolerance in rodents with MI.^125,132,133 In comparison, HIIT on a treadmill elicits similar^125,132 or even better effects¹³³ to those of moderate-intensity aerobic exercise. However, it is noteworthy that HIIT (with a 15° incline) may cause left ventricular dilation and cardiac mitochondrial injury in animals with MI injury.¹³²

Additionally, HIIT can be used to induce physiological cardiac hypertrophy, which is a beneficial adaptation of the heart to exercise. For instance, HIIT consisting of 8 min of continuous high-intensity running (85%–90% VO_2max) and 2 min of interval running (50%–60% VO_2max) on a slope of 25°, and lasting 1–2 h/day for 8 weeks, induces physiological cardiac hypertrophy in rats.^130,140

2.2.4.2. Swimming

Swimming training is another commonly used aerobic exercise mode in animal experiments. After allowing the rodents to acclimate to the aquatic environment for 5–10 min on the first day, it is recommended to begin adaptation training with 10 min of swimming, once or twice daily.^25,28,141 This duration is then progressively increased to 45–90 min per session over a period of 5–8 days.^25,28,141 Subsequently, the formal training protocol typically involves swimming for 45–90 min per session, once or twice daily, for a total duration of 3–16 weeks.^113,128,134 In a study using ApoE^-/- mice, the swimming exercise program was designed as 45 min/day, 3 days/week, for 8 or 16 weeks. This training suppressed immune cell accumulation in the fatty streak lesions at 8 weeks and reduced fibrofatty plaques at 16 weeks.¹²⁸ A swimming program with 90 min/session, twice a day, for 3–4 weeks in mice,¹⁴¹ or with 60 min/session, twice a day (with a 3% BW load from the third day), for 8 weeks in rats,²⁵ can alleviate myocardial I/R injury and heart failure.

Swimming exercise is also commonly used to induce physiological cardiac growth in rodents. Adult C57BL/6 mice that swim for 90 min, twice a day for 4 weeks (inclusive of the adaptation training) display an increase of heart weight-to-body weight (HW/BW) ratio and heart weight-to-tibia length (HW/TL) ratio.^28,142 A swimming program of 60 min/day, twice a day for 8 weeks (an additional overload of 3% BW added from the third day) can also induce physiological cardiac hypertrophy in rats.²⁵ In addition to physiological cardiac hypertrophy, these swim-trained mice demonstrate increased proliferative capacity of cardiomyocytes. The molecular mechanisms underlying exercise-induced physiological cardiac growth are important for blocking bad hypertrophy and promoting cardiac repair after cardiovascular injuries.²⁸

2.2.4.3. Voluntary wheel running

A voluntary wheel running program lasting 4–15 weeks can significantly improve CAD in animal models.^135,143,144 To explore the effect of voluntary running on cardiac function in mice, metal wheels with a diameter of 11.5 cm are placed into cages that are 47.0 cm long × 26.0 cm wide × 14.5 cm high.³⁸ In rat studies, a 35-cm-diameter running wheel is attached to the home cage compartment to facilitate voluntary running.¹⁴⁵ The maximum running speed, total run distance, and total run time of rodents are recorded by digital counters connected with the wheel. The HW/BW ratio is increased after 2 weeks of voluntary running, and persists through 4 weeks of exercise.³⁸ It has been reported that mice having free access to a running wheel for 4 weeks presented an ameliorated systemic inflammatory state and a stabilization of vulnerable lesions during atherosclerosis development.¹⁴³ Another study demonstrated that rats with 15 weeks of voluntary wheel running had improved insulin resistance and reduced proliferation of vascular smooth muscle cells in a genetically spontaneous atherosclerosis model.¹⁴⁶

2.2.4.4. Intervention time

The timing of exercise intervention is designed according to the purpose of the study. To investigate the preventive and protective effects of exercise, exercise training interventions should be conducted prior to disease onset (e.g., before LAD surgery) or during disease development (e.g., during diet-induced pathogenesis).¹⁴⁷ In contrast, when evaluating the therapeutic effects of exercise on CAD, exercise rehabilitation process should commence after the establishment of disease models.¹⁴⁸ For example, to study the effects of exercise on the progression of atherosclerosis, running intervention is usually carried out at the same time as HFD start.^123,131 To study the therapeutic effect of exercise on MI, an exercise intervention is initiated after the surgical procedures that induce MI through LAD artery ligation.¹²⁵

2.2.5. Evaluation metrics in CAD research with an exercise intervention

Exercise demonstrates multiple beneficial effects on cardiovascular health through distinct mechanisms: it enhances myocardial survival, improves endothelial function, reduces vascular stenosis, and optimizes microvascular function and hemodynamics.^11,112 In the study using the exercise-induced physiological cardiac hypertrophy model, HW/BW ratio and HW/TL ratios are essential indices to evaluate the increased heart weight in response to exercise.^130,140,149 Wheat germ agglutinin staining for myocardial cross-sectional area, 5-ethynyl-2′-deoxyuridine (EdU) (or Ki67) and α-actinin immunofluorescent staining for cardiomyocyte proliferative activity, and expressions of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) (to exclude pathological cardiac hypertrophy) are essential for investigating the effect of exercise on promoting the physiological hypertrophy and proliferative capacity of cardiomyocytes.

To study the effects of exercise in models of atherosclerosis, the impact of exercise on vascular function is pivotal; it can be tested by evaluating whether exercise could improve the in vitro vasodilation capacity of blood vessels.^113,122 Other detection indexes include the characteristics of plaque features, lipid metabolism level, and coronary arterial stiffness.^131,134,150 Coronary arterial function can be measured by the response to a series of concentrations of vasoactive agents, such as acetylcholine (ACh), NO (diethylamine-NONOate), or sodium nitroprusside.^113,122 Coronary arterial stiffness can be measured by making a stress–strain curve through a Multi Wire Myograph System, by using atomic force microscopy, or by measuring in vivo pulse wave velocity.^150,151

When the effect of exercise is evaluated in the MI or myocardial I/R model, cardiac function as measured by echocardiography is critical. The improved cardiac systolic function (e.g., left ventricular ejection fraction and fractional shortening) after exercise compared to the non-exercise group is an important indicator of the beneficial effect of exercise. The terminal deoxynucleotidyl transferase 2′-deoxyuridine 5′-triphosphate (dUTP) nick end labeling and α-actinin staining of cardiac tissues are used to determine whether exercise can reduce myocardial apoptosis. Other common monitoring indicators include the myocardial infarct size, as measured by TTC staining, and cardiac fibrosis as measured by Masson’s trichome staining.^113,121 The levels of SOD, malondialdehyde, nitrotyrosine, and inflammatory factors can be measured to evaluate the oxidative stress and inflammation responses in the heart.^131,134 The main detection index for evaluating exercise-induced effects on CAD are summarized in the following table (Table 2).

Table 2.

Evaluation metrics in CAD research that could be examined with an exercise intervention.

Detection index	Parameters	Reference
Body weight and heart weight	BW, TL, HW, HW/BW ratio, HW/TL ratio These parameters should also be measured in studies of exercise-induced physiological cardiac hypertrophy	130,140,142,149
Cardiac function	Echocardiography	113,121
Blood lipid	TC, TG, LDL, HDL levels detected by biochemical analyzer	131,134
Myocardial infarct size	TTC staining	126,136
Arterial function assessment	Responses of coronary arteries to ACh, NO (diethylamine-NONOate), or sodium nitroprusside	113,122
Arterial stiffness test	Calculated by making a stress–strain curve through a Multi Wire Myograph System Stiffness of endothelial cells within intact aortic segments by atomic force microscopy In vivo pulse wave velocity measured by a doppler ultrasound	150,151
Plaque features	Plaque area, plaque rupture rate, and vulnerable index Coronary arterial diameter and wall thickness are assessed by histological sections Staining of α-smooth muscle actin in coronary arterial walls	131,134
Cardiomyocyte features	WGA staining, EdU (or Ki67) and α-actinin staining	25,28
Cell apoptosis and cardiac fibrosis	TUNEL staining, Western blotting for Bax, Bcl-2, and Caspase3 cleavage Masson’s trichrome staining, Western blotting or qRT-PCR for Col1a1, Col3a1, and α-SMA	126,136
Oxidative stress	SOD, MDA, nitrotyrosine, etc.	131
Inflammation responses	Levels of CXCL1, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17, TNF-α, TGF-β, MMP-9 and its endogenous inhibitor TIMP1, etc.	131,134,135

Abbreviations: α-SMA = alpha-smooth muscle actin; ACh = acetylcholine; Bax = Bcl-2-associated X, apoptosis regulator; Bcl-2 = B-cell lymphoma 2; BW = body weight; CAD = coronary artery disease; Col1a1 = collagen type I alpha 1 chain; Col3a1 = collagen type III alpha 1 chain; CXCL1 = chemokine (C-X-C motif) ligand 1; EdU = 5-ethynyl-2′-deoxyuridine; HDL = high density lipoprotein; HW = heart weight; IL = interleukin; LDL = low density lipoprotein; MDA = malondialdehyde; MMP-9 = matrix metalloproteinase-9; NO = nitric oxide; qRT-PCR = quantitative real-time polymerase chain reaction. SOD = superoxide dismutase; TC = total cholesterol; TG = triglyceride; TGF-β = transforming growth factor-β; TIMP1 = Tissue inhibitor of metalloproteinases 1; TL = tibia length; TNF-α = tumor necrosis factor-α; TTC = 2,3,5-triphenyltetrazolium chloride; TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling; WGA = wheat germ agglutinin.

2.2.6. Recommendations

• •Various exercise types, including treadmill running, swimming, and voluntary wheel running, showed the ability to improve CAD.
• •Moderate-intensity treadmill running is recommended to be performed with an intensity of 50%–70% VO_2max (12–20 m/min in mice,^121,122,131 16–25 m/min in rats^137,138) for 60–90 min/day, 5–7 days/week, for 4–12 weeks, depending on the functional changes of interest.
• •To investigate the effect of exercise in very early stage of atherosclerosis, moderate-intensity treadmill running (e.g., 15 m/min for 60 min/day, 4 weeks in total in LDL-R^-/- mice) is effective to reduce endothelial dysfunction and redox imbalance, despite the fact that total cholesterol and triglyceride levels were unaltered by exercise.¹²²
• •To attenuate atherosclerotic plaque growth, 8 weeks of moderate-intensity treadmill running (e.g., 15 m/min for 60 min/day, initiate running after 3 weeks on a HFD) can reduce atherosclerotic inflammation and enhance plaque stability in ApoE^-/- mice fed with a HFD, even without a marked reduction in BW and blood lipid levels.¹³¹ In comparison, 12 weeks of moderate-intensity treadmill running (e.g., 15 m/min for 60 min/day, initiate running after 4 weeks on a Western diet) can reduce BW (from the 10th week on a Western diet) and ameliorate blood lipid profiles and atherosclerosis.¹²³
• •HIIT in CAD research generally refers to running protocols that involve alternating between intervals of 4–8 min of running at high-intensity (80%–90% VO_2max) and 3–4 min of running at low-to-moderate intensity (40%–60% VO_2max) for about 50–60 min/day, for 4–8 weeks in rodents.^125,132,133 HIIT treadmill running can exhibit similar^125,132 or even better effects¹³³ in the improvement of exercise capacity and cardiac function in MI rodents.
• •To explore the preventive effect of swimming against I/R injury, swimming programs involving 90-min sessions twice a day for 3–4 weeks in mice,¹⁴¹ or 60-min sessions twice a day (with a 3% BW load from the third day) for 8 weeks in rats,²⁵ can decrease infarct size and alleviate myocardial I/R injury.
• •To induce physiological cardiac hypertrophy, swimming exercise of 90 min/session, twice a day, for 4 weeks (inclusive of an adaptation training) is recommended in mice; this can efficiently promote both myocardial hypertrophy and cardiomyocyte proliferative activity.^28,142 Alternatively, HIIT induces a hypertrophic response in cardiomyocytes.^130,152,153 When training is discontinued and a sedentary lifestyle is resumed, these training-induced adaptations return to baseline levels within 2–4 weeks. The loss of training-induced effects occurs more rapidly than the development of those effects.¹⁴⁰
• •Voluntary wheel running for 4 weeks in mice,^38,154 or 6–8 weeks in rats,^155,156 can induce mild cardiac growth. Voluntary wheel running for a minimum of 4 weeks can alleviate atherosclerosis¹³⁵ and MI.¹²⁶

3. Animal exercise intervention studies for metabolic diseases

3.1. Obesity

3.1.1. Introduction to obesity

Obesity is a chronic disease marked by an abnormal or excess accumulation of body fat, and it is generally defined by a BMI of 30 kg/m² or higher. According to the epidemiological data from WHO, global obesity prevalence has more than doubled since 1990; over 890 million (16%) adults were obese in 2022, which poses a major health and economic burden to all societies.¹⁵⁷ In line with the global trend, obesity is also highly prevalent in China. Data from the China Chronic Disease and Risk Factors Surveillance program suggest that the prevalence of adult obesity in China had more than doubled between 2004 (3.1%) and 2018 (8.1%), with an estimated 85 million individuals who were obese in 2018.¹⁵⁸

Obesity is primarily caused by a long-term imbalance between energy intake and energy expenditure, with a tendency for the former to exceed the latter. It is a complex disease stemming from the intricate interplay of genetic, metabolic, behavioral, and environmental factors. Furthermore, obesity poses a significant risk factor for many metabolic disorders, such as diabetes, hypertension, cardiovascular diseases, fatty liver diseases, and several cancers (e.g., breast, liver, and colorectal cancers).159, 160, 161, 162, 163

3.1.2. Epidemiological studies of exercise intervention in obesity

Lifestyle interventions, including dietary intervention and exercise, are the first line of action and remain foundational to manage obesity and obesity-related conditions even when more interventions, like drugs or surgery, are required.164, 165, 166 Notably, exercise is helpful in the management of obesity and can play a key role in BW maintenance and health promotion.167, 168, 169 At least 150–300 min per week of moderate physical activity is recommended for weight maintenance and overall health promotion.170, 171, 172 Based on evidence from published systematic literature reviews, moderate-intensity aerobic exercise is recommended for reducing BW, total fat and visceral fat, and for improving blood pressure as well. Meanwhile, resistance training at moderate-to-high intensity is recommended for preserving lean mass during weight loss. Engaging in various forms of exercise (aerobic, resistance, and combined aerobic–resistance) or HIIT (after a comprehensive cardiovascular risk assessment and under supervision) is advised for improving insulin sensitivity and increasing cardiorespiratory fitness.173, 174, 175 Furthermore, for adults who have severe obesity (BMI ≥ 40.0 kg/m² or 35.0–39.9 kg/m² accompanied by at least one obesity-related comorbidity), long-term combination of moderate-intensity and HIIT aerobic exercise appears to be more efficacious in facilitating weight loss compared to moderate-intensity exercise alone.¹⁷⁶

It is important to note that exercise confers multiple health benefits for overweight and obese individuals, even in the absence of weight loss.^177,178 While the impact of exercise on weight loss might be limited, engaging in regular exercise has been proven effective at decreasing excess fat—particularly visceral adipose tissue, which is commonly a significant contributor to an increased risk of developing metabolic syndrome.¹⁷⁹ Evidence shows that multiple types of exercise (e.g., aerobic exercise performed at least at moderate intensity, resistance training, combined aerobic and resistance training, and HIIT) are beneficial in reducing visceral adipose tissue among individuals who are overweight or obese.¹⁸⁰ In addition, high-intensity aerobic exercise and HIIT appear to be the most effective exercise interventions for decreasing visceral adipose tissue, BW, total body fat, BMI, waist circumference, and subcutaneous adipose tissue.¹⁸⁰ For older adults with obesity, a dietary intervention combined with both aerobic and resistance exercise has proven to be more effective at reducing intermuscular and visceral adipose tissue and improving physical and metabolic function when compared to a dietary intervention paired with either aerobic or resistance exercise alone.¹⁸¹ Moreover, physical fitness plays an important role in promoting health and is widely recognized as an indicator of overall well-being.¹⁸² Regular exercise also improves physical fitness in overweight or obese individuals, including cardiorespiratory fitness, muscle strength, and motor capacities.^183,184 Furthermore, obesity is typically accompanied by chronic low-grade inflammation in multiple organ systems, such as adipose tissue, liver, skeletal muscle, heart, pancreas, and brain; and the activation of inflammation is associated with increased risk of metabolic syndrome in adults with obesity.¹⁸⁵ A great body of evidence shows that exercise exerts beneficial effects on systemic inflammation. One systematic review shows that long-term endurance, resistance, or HIIT exercise can reduce levels of circulating pro-inflammatory cytokines, including IL-6, C-reactive protein (CRP), and TNF-α, whereas resistance exercise can also increase levels of anti-inflammatory cytokines such as IL-10.¹⁸⁶

3.1.3. Basic research on the effect of exercise in obesity

Despite exercise having long been known for its role in treating obesity, the underlying mechanisms are complex. Exercise can help reduce systemic inflammation and improve overall metabolism, especially insulin sensitivity in obese adults.^180,186, 187, 188, 189 Exercise can also promote the health outcomes of obesity by improving gut health, increasing microbial diversity and abundance, and altering neurotransmitters that regulate appetite.¹⁹⁰ The alteration of adipose tissue is a critical mechanism underlying the benefits of exercise.¹⁹¹ On one hand, exercise can increase fat oxidation and reduce body fat in obese individuals, which may be facilitated through the regulation of adipose tissue lipolysis and modulation of adipocytokine gene expression.¹⁹² On the other hand, exercise can improve obesity-induced adipose tissue inflammation partly via decreasing immune cell infiltrations and possibly by reducing “inflammatory biased” immune cells.¹⁹³ However, the detailed molecular mechanisms of the effect of exercise on obesity are still obscure and need further investigation.

3.1.4. Animal exercise intervention studies and models for obesity

Currently, most studies on obesity utilize a model established through the administration of a HFD feeding for 3–4 months or even longer. In studies examining the effect of exercise on obesity, 3 types of exercise are commonly used: treadmill running, swimming, and voluntary wheel running (Supplementary Table 3). In general, all 3 types of exercise are effective in promoting weight and fat loss as well as improving metabolism in obese animals, including decreased fasting insulin level, reduced serum triglyceride (TG) and total cholesterol (TC) levels, and improved glucose homeostasis and insulin sensitivity.194, 195, 196, 197, 198, 199 Moreover, long-term exercise also confers protection against obesity-related disorders. There is evidence showing that 11 months of voluntary wheel running can significantly attenuate obesity-induced cognitive impairment and white matter damage in mice.²⁰⁰ However, the health benefits of exercise can vary depending on the intensity and duration of the training. For example, evidence shows that while both moderate treadmill exercise and HIIT are effective for reducing BW and fat mass, HIIT is particularly beneficial for inhibiting fat accumulation.^194,201 Furthermore, another study has reported that both moderate-intensity exercise and HIIT can equally enhance aerobic capacity and reduce obesity, yet HIIT stands out as superior in improving glucose tolerance.²⁰² Additionally, a recent study reported that all 3 intensities (low, moderate, and high) of treadmill exercise prevented obesity and metabolic syndrome in HFD-fed mice.²⁰³ However, both moderate- and high-intensity exercise impaired cardiac function in HFD-fed mice, whereas low-intensity exercise improved cardiac health.²⁰³ It is also noteworthy that the running capacity of obese animals is observed to be lower than that of control animals.^198,202 Exercise can counteract the obesity-induced decrease in running capacity, restoring it to levels comparable to those of control animals.^198,202

There appear to be sex differences in the protective effect of exercise against obesity. The effect of exercise on obesity in different sexes is complex and requires further investigations. One study reports that although female rats are generally more willing to run voluntarily, exercise appears to confer greater benefits in male rats in terms of reducing HFD-induced weight gain and fat accumulation as well as improving glucose metabolism.²⁰⁴ Conversely, another study found that 8 weeks of treadmill exercise reduced weight gain and improved glucose tolerance in female mice but not males.²⁰⁵ These discrepancies may partly arise from differences in exercise type and intensity across studies. Further studies are needed to identify and elucidate the sex differences in exercise-mediated protection against obesity.

3.1.4.1. Treadmill running

Low-intensity: Limited studies have investigated the effects of low-intensity treadmill running on obesity. A study reports that mice running at 6 m/min for 1 h on a 5° slope 5 days/week for 8 weeks significantly prevents HFD-induced weight gain.²⁰³ Notably, this training program also confers protection against HFD-induced impairment in cardiac function, which is, however, aggravated by continuous moderate- and high-intensity treadmill exercise.²⁰³

Moderate-intensity: Moderate-intensity treadmill running has been widely used in obesity studies. Commonly, these training regimens include 5–7 days of adaptive training before regular treadmill running. For adaptive training, animals (rats or mice) can start running at 5 m/min for 10 min per day with a gradually increased speed and duration.^201,206,207 The detailed exercise patterns, such as running speed, frequency, and duration, vary depending on the research purpose and the severity of obesity. More significant BW loss and additional health benefits, such as reduced steatosis and improved systemic inflammation and cardiac function, can be observed with a longer intervention time.^201,202,207

To study the preventative effect of moderate-intensity treadmill running on obesity, the training regimens can be initiated when the HFD is introduced or during the early development of obesity. Mice can run at a speed of 12–17 m/min for 45–60 min/day on a 0°–10° slope with a frequency of 5 days/week.^203,206, 207, 208 Of note, an incline is not necessary for studying the effect of exercise on obesity. However, moderate exercise with a higher speed, longer daily duration, and steeper incline seems to offer greater benefits in terms of BW loss.^207,208 When exercise intervention begins simultaneously with the introduction of a HFD, the BW gain decreases soon after starting the exercise.²⁰⁷ In contrast, when exercise intervention starts during the development of obesity (e.g., 4 weeks after the implementation of a HFD), an obvious reduction in BW can be observed as early as after 2 weeks of exercise.²⁰⁸ It is noteworthy that running at a moderate intensity with a shorter daily duration (e.g., 12 m/min for 30 min/day with a frequency of 5 days/week in mice) may exert no significant change on BW even after 12 weeks of training. However, it can beneficially affect the oxidative capacity in the skeletal muscle of obese mice.²⁰⁶

To study the therapeutic effect of moderate-intensity exercise on obesity, training regimens can be started after the establishment of obesity. Specifically, when mice run at a moderate intensity (65%–70% VO_2max) for 90–120 min/day at a 25° slope with a frequency of 5–6 days/week, a significant reduction in BW can be observed as early as after 2 weeks of exercise.^201,202 Alternatively, when rats run at 15–20 m/min (start at 15 m/min and gradually elevate to 20 m/min during the running program) for 30–90 min/day (start at 30 min and gradually elevate to 90 min) with a frequency of 6 days/week, a significant reduction in BW can be observed as early as after 1 week of exercise.¹⁹⁵

High-intensity: Compared to moderate-intensity treadmill exercise, high-intensity training appears to yield superior health outcomes in obese animals, especially through better weight maintenance¹⁹⁴ and more favorable metabolic benefits, such as reduced lipid accumulation in the liver and improved systemic inflammation.^201,202 When engaging in high-intensity exercise, it is advisable for animals to start with a lower speed and then gradually increase the running speed.²⁰⁹ For example, mice can begin with a warm-up run at 5 m/min for 10 min before starting the HIIT session.²⁰¹ Then they can run at 85%–90% VO_2max for 4 min, completing 10 bouts, with each bout followed by 2 min of active rest at 5 m/min.^201,202 The total duration of each training session, including rest periods, is almost 1 h per day, with the frequency set at 5 days/week.^201,202 Soon after starting the exercise, the BW gain of exercised obese mice decreases compared to that of sedentary obese mice, with more significant BW loss observed as the intervention time increases.²⁰¹ Alternatively, animals can run at high intensity (mice at 18 m/min²⁰³ and rats at 25–30 m/min²⁰⁹) for 60 min/day, 4–5 days/week. However, it is worth noting that although continuous high-intensity running is effective in preventing obesity and metabolic dysfunction, it may be harmful to the heart.²⁰³ Additionally, for severe obesity (where the BW of obese animals is almost double that of control animals), a training regimen consisting of running at 15 m/min for 15 min, then at 25 m/min for 50 min, 5 days/week for 3 weeks can significantly prevent BW gain in the HFD-induced obese rats.²¹⁰ More significant BW loss can be observed with longer intervention times.

3.1.4.2. Swimming

Swimming training is another widely used exercise type in obesity studies. For swimming exercise, training can be conducted in a tank containing warm water with the temperature controlled at 30°C–32°C.211, 212, 213, 214 Swimming training begins with 2–7 days of acclimatization training. During the initial training period, animals can swim freely with the duration progressively increased from 10−20 min to 60–120 min.^196,197,212, 213, 214, 215 Evidence shows that 60–120 min of swimming exercise per day, 5 days/week for 8–16 weeks prevents or attenuates obesity and improves obesity-related conditions, including cognitive function, vascular health, and reproductive function.^196,197,211, 212, 213^,216 Of note, training intensity can be elevated during the swimming program by attaching a tail weight equivalent to 5% of the animal’s BW.²¹² The swimming exercise model provides a low-cost way to standardize the dose of exercise (intensity and duration) across strains or genetic models that may have different voluntary thresholds for exercise.

3.1.4.3. Voluntary wheel running

In terms of voluntary wheel running, both mice and rats are commonly used in the exercise intervention studies of obesity. Animals are usually individually housed and have free access to running wheels. To conduct voluntary wheel running, mice can be housed in a polycarbonate cage (20.5 cm wide × 36.5 cm long × 14.0 cm high) with a running wheel (wheel diameter of 10.2 cm, interior diameter of 9.2 cm, wheel width of 5.1 cm),¹⁹⁹ and rats can be provided with a running wheel with a diameter of 36.0 cm and a width of 11.0 cm.¹⁹⁸ Assessment of running distance, average speed, maximum speed, and running time are generally needed for the analysis of running capacity.^198,199,217, 218, 219 In both preventative and therapeutic studies of exercise, a reduction in BW can be seen shortly after starting the voluntary wheel running, and this loss becomes more pronounced as the intervention continues.^198,199 However, a preventative study reported that significant weight loss appeared after 10 weeks of voluntary training and persisted until the end of the training period.²¹⁹ The discrepancy may partly be explained by differences in the HFD and its duration as well as the age of the animals. It should also be noted that a 6-week voluntary wheel running program can significantly reduce body fat mass in obese mice even without significant BW change.²¹⁸ Additionally, to prevent obesity-related cognitive impairment, voluntary wheel running exercise can be started 1 month prior to the HFD feeding and the duration of exercise can be set to 11 months.²⁰⁰

3.1.4.4. Intervention time

Typically, animals are administered a HFD that contains 45%–60% of calories from fats to establish models of obesity. In contrast, mice fed a control/standard chow diet that contains 3.5%–16.0% of calories from fats serve as control subjects.^{199,201,211,212,216,}218, 219, 220 Animals fed the HFD are deemed obese if their BW exceeds 20% of the average weight of the control group,^195,213 with the corresponding feeding time commonly being 16–20 weeks.^210,221, 222, 223 Alternatively, animals with a marked increase in body fat, typically exhibiting a doubling of fat mass relative to controls, are considered obese.²¹⁸ However, it is noteworthy that the metabolic changes typical of obesity, such as impaired glucose tolerance, can be observed as early as 1 week after HFD feeding and become apparent by 8–12 weeks.²²¹ Therefore, to study the effects and the underlying mechanisms of exercise on obesity, the timing of intervention depends on the specific research goal of the study and the severity of obesity required. Moreover, as nutritional composition is closely associated with the development of obesity, the control/standard chow diet should only differ from the HFD in terms of the composition of carbohydrates or fats.²²² This helps to translate the findings in an unbiased manner. In addition, HFD feeding usually continues even after the establishment of obesity, until the end of the animal experiments.

3.1.5. Evaluation metrics in obesity research with an exercise intervention

To evaluate the effect of exercise on obese animals, parameters related to obesity phenotypes should be evaluated. These include BW and body fat mass changes as well as metabolic disturbances such as glucose intolerance and insulin resistance. BW should be analyzed weekly. Moreover, obesity is generally associated with increased levels of plasma glucose or fasting blood glucose, higher levels of plasma insulin or fasting insulin, as well as elevated levels of plasma TG and TC, which should also be analyzed (Table 3). Note that for HFD-induced obesity, food intake is generally required to be recorded during the entire experiment. Furthermore, evidence suggests that obesity is linked to a decline in cognitive function,^200,211 disrupted muscle metabolism,²⁰⁶ compromised vascular function,¹⁹⁷ and reproductive dysfunction.²¹³ Thus, the analysis of obesity-related disorders may also reflect the preventative or therapeutic effect of exercise on obesity.

Table 3.

Evaluation metrics in obesity research that could be examined with an exercise intervention.

Detection index	Parameters	Reference
Body weight	Exceeds 20% of the average weight of control animals	195,213
Fat mass	A doubling of fat mass relative to controls	218
Blood glucose level	Increased blood glucose or fasting blood glucose level	198,207
Blood lipid content	Increased levels of blood TG and TC	195,197,201
Blood insulin level	Increased blood insulin or fasting insulin level	195,219
Glucose intolerance	Glucose tolerance test	196,201,202
Insulin resistance	Insulin tolerance test, HOMA-IR	195,196,201

Abbreviations: HOMA-IR = homeostasis model assessment of the insulin resistance; TC = total cholesterol; TG = triglyceride.

3.1.6. Recommendations

• •All 3 exercise types—treadmill running, swimming, and voluntary wheel running—are effective for weight and fat loss195, 196, 197, 198, 199 and are commonly used in the obesity studies.
• •To prevent obesity via treadmill exercise, mice can run at 12–17 m/min for 45–60 min/day on a 0°–10° slope for 8–12 weeks.^203,206, 207, 208 The training regimens can be initiated when the HFD is introduced or during the early development of obesity.
• •To treat obesity via treadmill exercise, animals can run at a moderate intensity (65%–70% VO_2max) for 90–120 min/day (mice) or 30–90 min/day (rats) on a 0°–25° slope with a frequency of 5–6 days/week.^195,201,202 For better weight maintenance and more favorable metabolic benefits, animals can undergo HIIT exercise (interval 85%–90%VO_2max) for 1 h per day, with a frequency of 4–5 days/week.^201,202,209
• •An incline is not necessary for studying the effect of treadmill running on obesity. However, moderate exercise with a higher speed, longer daily duration, and steeper incline seems to offer greater benefits in terms of BW loss.^207,208
• •To reduce obesity via swimming exercise, animals can swim 60–120 min per day with a frequency of 5 days/week for at least 3–5 weeks.^196,197,212, 213, 214, 215
• •To reduce obesity via voluntary wheel running, a reduction in BW can be seen shortly after starting the exercise, with more significant BW loss observed as the intervention time increases.^198,199,215
• •The duration of exercise intervention depends on the specific research goal of the study and the severity of obesity. Generally, more significant BW loss and additional health benefits can be observed with a longer intervention duration.^{196,198,199,201,202,207,211,216,219}

3.2. T2DM

3.2.1. Introduction to T2DM

T2DM is one of the most common chronic metabolic disorders, characterized by elevated blood glucose levels and insulin resistance and commonly associated with complications in peripheral organs, including the cardiovascular system, brain, liver, kidneys, and retina.^224,225 The estimated number of people living with diabetes globally was 536.6 million in 2021, with the majority being T2DM, and this number is projected to rise to 783.2 million by 2045.²²⁶ It should be noted that China is one of the most prevalent countries for T2DM.²²⁷

The main pathogenesis of T2DM is insulin resistance in peripheral tissues and deficiency of insulin secretion in pancreatic β-cells.^228,229 Defects in the pathways involved in the synthesis and release of insulin or the insulin response in tissues can lead to the development of T2DM.²²⁸ Key factors contributing to hyperglycemia include gut microbiota imbalances, inflammation, immune dysregulation, incretin dysregulation, increased kidney glucose reabsorption, central appetite dysregulation, islet amyloid polypeptide (amylin) deposition in the pancreas, and insulin resistance in the liver, muscle, and adipose tissue.^230,231

3.2.2. Epidemiological studies of exercise intervention in T2DM

The prevalence of T2DM has been largely attributed to changes in living environments and lifestyles, including diet and physical activity levels.²³² Much evidence suggests that many cases of T2DM can be prevented by improving the main modifiable risk factors, such as obesity, physical inactivity, and unhealthy diet.233, 234, 235 It is well known that exercise can enhance health and manage blood glucose level in individuals with T2DM. Generally, exercise is an essential component in the prevention and management of T2DM.²³⁶ Advice on engaging in regular exercise is always included in the management of T2DM.²³⁷ A consensus statement from the American College of Sports Medicine recommends that various types of exercise, such as aerobic exercise, resistance exercise, flexibility exercise, and balance exercise, can be applied to individuals with T2DM.²³⁸ Long-term aerobic exercise can improve glycemic management in diabetic individuals, with less daily time in hyperglycemia and 0.5%–0.7% reductions in overall glycemia.²³⁸ High-intensity resistance exercise has been shown to be more effective than low-to-moderate-intensity resistance exercise in terms of overall glucose management and attenuation of insulin levels.²³⁸ Moreover, combined aerobic and resistance training seems to be the optimal form of exercise prescriptions for T2DM.^237,239 While exercise is considered a fundamental strategy for T2DM, its benefits are not experienced uniformly across all individuals.²³⁹ Therefore, it is essential to intensify research into the mechanisms underlying the effects of exercise and to determine the suitable type, dosage, and intensity of exercise prescriptions. Animal exercise models can serve as a valuable tool in this exploration.

3.2.3. Basic research on the effect of exercise in T2DM

Long-term exercise is well known for its beneficial role in controlling blood glucose and improving prognosis of T2DM. The underlying mechanisms of exercise in regulating glucose and lipid metabolism involve the increase of glucose uptake and utilization, improvement of insulin sensitivity, enhancement of pancreatic β-cell function, increase of lipid hydrolysis, and attenuation of systemic inflammation, as well as the secretion of cytokines such as irisin, osteocalcin, and adiponectin.²⁴⁰ Exercise can also improve diabetes and its complications via the alleviation of endoplasmic reticulum (ER) stress through decreasing oxidative stress, maintaining the stability and function of mitochondria-associated ER membranes, and reducing inflammation levels.²³⁹ Additionally, there is evidence showing that exercise alleviates T2DM via the enhancement of mitochondrial function; the possible mechanisms are linked to enhanced mitochondrial biogenesis via activation of adenylate activated protein kinase (AMPK)/peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signaling and increased mitophagy.²⁴¹

3.2.4. Animal exercise intervention studies and models for T2DM

HFD with streptozotocin (STZ) injections or db/db mice are often used as animal models for T2DM. In the current studies on T2DM, a variety of exercise types are employed, including treadmill running, swimming, voluntary wheel running, and resistance exercise. For example, evidence shows that mice running at an intensity of 7–11 m/min for 1 h per day, 5 days/week for 8 weeks can effectively ameliorate T2DM and its related complications, as evidenced by decreased blood glucose, insulin, and glycated hemoglobin A1c (HbA1c) levels, as well as improved glucose intolerance, reduced oxidative stress, and decreased bone loss in T2DM mice.²⁴² Moreover, a 24-week HIIT program has been shown to significantly improve T2DM and cardiac function.²⁴³ As treadmill running, voluntary wheel running, and swimming training are well-established and widely used in experimental T2DM models, here we mainly focus on these 3 types of exercise. It should be noted that, in terms of exercise capacity, diabetic animals exhibit significantly lower running speeds compared to age-matched healthy controls.²⁴⁴ While VO_2max remained unchanged in 6-week-old and 12-week-old db/db mice compared to age-matched wild-type (WT) controls, the maximum running speed (observed running speed at which VO_2max or peak oxygen uptake (VO_2peak) was achieved) of db/db mice was significantly lower: 34.6% less at 6 weeks old (36 ± 1 m/min for WT mice and 24 ± 1 m/min for db/db mice) and 64.1% less at 12 weeks old (38 ± 1 m/min for WT mice and 13.8 ± 0.9 m/min for db/db mice).²⁴⁴ Additionally, the sex of animals could affect the effect of exercise. However, there seem to be relatively few studies reporting the sex differences in exercise-mediated protection against T2DM. One study reports that while T2DM induces cognitive impairment more severely in males than in female rats, an 8-week HIIT program preferentially rescues this condition in males.²⁴⁵ Mechanistically, the sex-specific benefits of exercise may be mediated by differences in adiponectin receptor expression and its downstream signaling pathways.²⁴⁵

3.2.4.1. Treadmill running

Treadmill running is one of the best-established exercise models in the study of T2DM. Current studies indicate that running at a low intensity (30% VO_2max) for 6 weeks, moderate intensity (50%–70% VO_2max) for 4–12 weeks, or high intensity (85%–90% VO_2max) for 8–24 weeks can effectively lower blood glucose levels in diabetic animals, such as mice or rats. The specific details regarding running intensity, frequency, and duration are outlined in Supplementary Table 4. Interestingly, there is evidence showing that running with a lower intensity (at a speed of 10 m/min or 15 m/min, equivalent to 30% VO_2max and 50% VO_2max, respectively) is more effective at controlling the blood glucose levels of diabetic rats than running with a higher intensity (20 m/min, equivalent to 70% VO_2max).²⁴⁶ However, another study reports that high-intensity interval treadmill exercise resulted in a more remarkable glucose-lowering effect in T2DM mice.²⁴³ This discrepancy may be partly attributed to the different types of animals and the variations in their age. Additionally, long-term high-intensity interval treadmill exercise has been shown to be superior to moderate-intensity treadmill exercise at ameliorating the onset and progression of diabetic heart disease in mice.^243,247 Moderate-intensity treadmill running failed to protect the heart if exercise was initiated after the onset of cardiac dysfunction in diabetic mice.²⁴⁷

Low-intensity: Relatively few studies have investigated the effects of low-intensity treadmill running on T2DM. Existing evidence shows that running for 1 h at a speed of 10 m/min (equivalent to 30% VO_2max), 5 days/week for 6 weeks can significantly decrease fasting blood glucose levels and improve insulin resistance in diabetic rats.²⁴⁶ A similar training regimen, consisting of 1 h of running at 9 m/min, 5 days/week for 6 weeks, can significantly enhance cardiac function in diabetic mice.²⁴⁸ This is associated with improved mitochondrial morphology and function as well as decreased acetylation of cardiac mitochondrial enzymes.²⁴⁸ Thus, when engaging in low-intensity treadmill exercise, diabetic animals can run at 30% VO_2max for 1 h, 5 days/week for 6 weeks to ameliorate the symptoms of T2DM.

Moderate-intensity: In the T2DM disease models, treadmill exercise at a moderate intensity is most studied. Accumulating evidence shows that a training regimen consisting of 1 h of running at a speed of 7.0–13.3 m/min per day, 5 days/week for 8–12 weeks can significantly decrease the symptoms of diabetes and improve diabetes-related conditions, including preventing bone loss, improving cardiac function, and enhancing cognitive function in diabetic mice.^242,247,249, 250, 251 Moreover, for diabetic rats, a training regimen consisting of 1 h of running at 15–20 m/min (equivalent to 50%–70% VO_2max) per day, 5 days/week for 6–8 weeks can significantly decrease blood glucose levels and improve diabetic cardiomyopathy.^246,252 Note that diabetic rats running at a lower speed is associated with decreased fasting blood glucose, fasting insulin, glycosylated serum protein, and insulin resistance index, suggesting better control of blood glucose levels.²⁴⁶ Furthermore, there is evidence showing that rats running at a gradually increased speed (15–20 m/min) and duration (0.5–1.5 h/day), 6 days/week for 4 weeks significantly reduced the symptoms of diabetes.¹⁹⁵ Therefore, to improve T2DM via moderate-intensity treadmill running, diabetic animals can run at 50%–70% VO_2max for 1 h per day, 5 days/week for 6–12 weeks. Alternatively, diabetic rats can run at a gradually increased speed (15–20 m/min) and duration (0.5–1.5 h/day), 6 days/week for 4 weeks. In addition, a treadmill training regimen conducted at a moderate intensity for 3 days/week for 12 weeks, with the duration increasing by 10 min/week until it reaches 60 min, can effectively prevent the development of T2DM and improve cognitive function in adult mice.²⁵³

High-intensity: When engaging in high-intensity treadmill exercise, a 24-week HIIT regimen including nine sets of 1.5 min high-intensity running sessions (85% VO_2max), with 1 min of active rest (running at a speed equivalent to 45% VO_2max) between sets, has been shown to significantly decrease fasting blood glucose level and improve cardiac function in diabetic mice when performed 5 days/week.^243,254 Similarly, an 8-week HIIT regimen consisting of four sets per day of 7 min of running at 25 m/min and 3 min at 15 m/min also resulted in significant reductions in fasting blood glucose levels, which is associated with increased abundance of short-chain fatty acid-producing intestinal bacteria.²⁵² Moreover, there is evidence showing a 4-week training regimen that includes 5–7 sets of 2 min of running at 80%–90% VO_2max, with recovery cycles at 30% VO_2max, performed at a frequency of 5 days/week can significantly decrease fasting blood glucose levels, increase serum insulin levels, and enhance cardiac function in diabetic rats.²⁵⁵ This is accompanied by upregulated messenger RNA (mRNA) levels of autophagy-related genes in the heart, including autophagy related 1 (ATG-1), autophagy related 5 (ATG-5), Beclin-1, and lysosome-associated membrane glycoprotein 2 (LAMP-2).²⁵⁵ Furthermore, an 8-week training regimen consisting of 10 bouts of 5 min of running at 85%–90% VO_2max, with 2 min of active rest (running at a speed equivalent to 45% VO_2max), on a 10° incline treadmill for 5 days/week can enhance cardiac function even without significant changes in blood glucose levels.^{202,220,247,254} In addition, studies have shown that an 8-week training regimen consisting of 10 sets of 4 min of running at 16–26 m/min (gradually increasing from 16 m/min to 26 m/min over 8 weeks, equivalent to 85%–90% VO_2max) interspersed with 2 min of running at 8 m/min on a 15° incline treadmill for 5 days/week improved glucose homeostasis and decreased lipid accumulation in the kidneys, yet induced renal fibrosis and injury in diabetic mice.^256,257

Thus, to ameliorate T2DM via high-intensity treadmill exercise, diabetic animals can run at 80%–90% VO_2max for 1.5–7.0 min, with 1.0−3.0 min active rest (equivalent to 30%–45% VO_2max) between sets, for 4–10 sets at a frequency of 5 days/week for 4–24 weeks. Note that slope is not necessary for studying the effects of running exercise on T2DM. The duration of training varies depending on research goals and the method of establishing the diabetic model. For example, for a diabetic model established by HFD feeding plus STZ injections, 8 weeks of training can significantly improve blood glucose levels,^252,257 and 24 weeks of training can improve both blood glucose levels and cardiac function.²⁴³ For db/db mice, 8 weeks of training can significantly enhance cardiac function.²⁴⁷ The decision to engage in high-intensity interval exercise should be carefully considered because while it may alleviate the symptoms of T2DM, it can also potentially lead to organ injury.^203,257

3.2.4.2. Swimming

Swimming exercise also significantly reduces blood glucose and improves insulin resistance in mice.258, 259, 260 Before the training, it is recommended that diabetic mice start with a week of adaptive training.258, 259, 260 For instance, mice can perform this adaptive swimming training for 10 min per day for 5 days.^258,259 Following this, the training can be performed for 30–60 min, 5 days/week for 3–6 weeks.258, 259, 260 Similarly, it is recommended that rats begin with an adaptive swimming training. Rats can perform this adaptive swimming training for, for example, 15 min per day for 5 days.²⁶¹ Then the training can be conducted for 30–60 min, 3 days/week for 8–11 weeks, which is effective to reduce blood glucose and improve insulin sensitivity in diabetic rats.^261,262

In addition to improving glucose intolerance in T2DM animals, swimming for 30–60 min, 5 days/week for 3–6 weeks also offers other health benefits for diabetic mice. These benefits include reduced systemic inflammation, mitigated ER stress in skeletal muscles, decreased brain oxidative stress, and alleviation of depression-like and anxiety-like behaviors.258, 259, 260 Moreover, for diabetic rats, swimming for 30 min, 3 days/week for 8 weeks can decrease inflammation markers in the heart.²⁶² Furthermore, 60 min of swimming training, 3 days/week for 11 weeks is associated with decreased systemic inflammation in diabetic rats, as indicated by reduced CRP and adiponectin levels.²⁶¹

3.2.4.3. Voluntary wheel running

Animals are usually individually housed in cages equipped with a rotating wheel for voluntary wheel running, with daily running activities recorded.²⁶³ To prevent the development of T2DM, the running can be started 2–13 weeks before the onset of diabetes^198,264, 265, 266, 267 and last for 16–22 weeks in mice²⁶⁷ and 1.5–17.0 months in rats.^198,264, 265, 266 In addition to improving hyperglycemia, 17 months of wheel running can also prevent bone deterioration and loss of grip strength in diabetic rats.^264,265 While most research has focused on the preventive effects of voluntary wheel running against T2DM, few studies have explored its therapeutic impact. Notably, even without improving hyperglycemia and glucose intolerance, initiating wheel running for 6 weeks after diabetes onset can protect the hearts of diabetic mice from oxidative stress and diastolic dysfunction.²⁶³ Additionally, 10 weeks of voluntary wheel running beginning after the confirmation of diabetes can prevent diabetes-related pulmonary complications and attenuate lung inflammation in diabetic rats.²⁶⁸

3.2.4.4. Intervention time

In general, to investigate the effect of exercise on the treatment of T2DM, it is recommended to start the exercise after confirming the establishment of the T2DM animal model. HIIT has been shown to have cardioprotective effects, while moderate-intensity running may fail to provide cardiovascular benefits when exercise is initiated after the establishment of cardiac dysfunction.^243,247 To prevent the development of T2DM, training can be initiated before the onset of T2DM. For example, for HFD feeding and STZ-induced T2DM models, preventive treadmill running exercise can be started when the HFD feeding is introduced.²⁵³

3.2.5. Evaluation metrics in T2DM research with an exercise intervention

The animal models of T2DM are mainly established by HFD feeding with STZ injections or by using db/db mice, which possess genetic deficiencies in the leptin receptor.^{247, 267,269−272} Generally, mice or rats are fed with a HFD containing 45% or 60% fat for a period of 4–12 weeks, after which they receive a single injection of STZ at a concentration of 30–100 mg/kg or multiple doses at a concentration of 20–50 mg/kg (Supplementary Table 4). The db/db mice develop hyperglycemia starting from the age of 8 weeks.²⁴⁷ Furthermore, animal models of T2DM can be established using TALLYHO/JngJ (TH) mice, Otsuka Long-Evans Tokushima Fatty (OLETF) rats, or Zucker Diabetic Fatty (ZDF) rats. Specifically, diabetes onset occurs around the age of 10 weeks in TH mice and the age of 18 weeks in OLETF rats.^267,270 Additionally, ZDF rats are fed with a Purina 5008 diet, a HFD, to establish the T2DM model.²⁶¹

Hyperglycemia is a defining characteristic of T2DM. In general, for mice and rats, a blood glucose level exceeding 250 mg/dL is indicative of diabetes.^271,272 Of note, for the HFD feeding and STZ injection-induced diabetes, animals that exhibit a fasting blood glucose level higher than 11.1 mmol/L at 3 days and 7 days after the last injection of STZ are considered diabetic.²⁶⁹ Moreover, diabetic animals are typically marked by increased fasting insulin levels, glucose intolerance, and insulin resistance. To evaluate the effects of exercise on T2DM, the detection index and related parameters listed in Table 4 should be analyzed.

Table 4.

Evaluation metrics in T2DM research that could be examined with an exercise intervention.

Detection index	Parameters	Reference
Blood glucose level	>250 mg/dayL or fasting hyperglycemia (>11.1 mmol/L)	260,263,271
Blood insulin level	Increased fasting insulin levels	242,253,269,271
Glucose tolerance	Glucose tolerance test	251,269
Insulin resistance	Insulin tolerance test, HOMA-IR	250,251,269
Blood glycated hemoglobin level	Increased blood glycated hemoglobin level	242

Abbreviations: HOMA-IR = homeostasis model assessment of the insulin resistance; T2DM = type 2 diabetes mellitus.

3.2.6. Recommendations

• •All 3 exercise types—treadmill running, swimming, and voluntary wheel running—can effectively reduce blood glucose levels and improve insulin resistance.^{242,246,259,261,267,268,271}
• •To decrease the symptoms of T2DM via treadmill exercise, diabetic animals can run at 30%–70% VO_2max for 1 h per day, 5 days/week for at least 6 weeks.^242,246, 247, 248, 249, 250, 251, 252
• •To achieve a significant reduction in blood glucose and improvement in insulin resistance, mice can engage in swimming exercise with a daily duration of 30–60 min, 5 days/week for 3−6 weeks.258, 259, 260 Similarly, rats can perform swimming with a daily duration of 30–60 min, 3 days/week for 8–11 weeks.^261,262
• •To prevent the development of T2DM, mice can engage in voluntary wheel running for 16–22 weeks²⁶⁷ and rats for 1.5–17.0 months.^198,264, 265, 266 The running can be started 2–13 weeks before the onset of diabetes.
• •HIIT seems to be superior to moderate-intensity running at improving cardiac function in diabetic aniamls.^243,247 However, the decision to engage in HIIT programs should be carefully considered because while it may ameliorate the symptoms of T2DM, it can potentially lead to organ injury.²⁵⁷

4. Animal exercise intervention studies for chronic obstructive pulmonary disease (COPD)

4.1. COPD

4.1.1. Introduction to COPD

COPD is a chronic lung condition caused by airway obstruction; it is characterized by progressive airflow limitation, airway remodeling, and lung emphysema.²⁷³ Symptoms of COPD include cough accompanied by phlegm, shortness of breath, wheezing, and fatigue due to abnormalities of the airway.²⁷⁴ In 2019, there were approximately 212.3 million prevalent cases of COPD worldwide, imposing a substantial economic burden and decreased health-related quality of life. Globally, deaths from COPD were 42.5 (95% uncertainty interval: 37.6–46.3) per 100,000 members of the population, leading to 3.3 million deaths in 2019.²⁷⁵ Cases of COPD are projected to approach 600 million globally by 2050.²⁷⁶ In China, data from a cross-sectional analysis of the China Pulmonary Health study, which was conducted on a nationally representative subset of adults aged 20 years and older, indicated that the estimated overall prevalence of spirometry-defined COPD was 8.6% (95% confidence interval (95%CI): 7.5–9.9) in 2012–2015. This corresponds to approximately 99.9 (95%CI: 76.3–135.7) million people affected by COPD in China.²⁷⁷ These data indicate that the disease has become a significant public health problem. Effective actions are required to strengthen prevention and management to reduce the disease burden caused by COPD.

Tobacco smoking is the leading cause of COPD, contributing to more than 70% of cases in high-income countries and 30%–40% of cases in low- and middle-income countries.²⁷⁸ Other risk factors include exposure to household or outdoor air pollution and occupational irritants like dust and chemicals. Early lung failure, childhood asthma, and inherited mutation (a rare α1 antitrypsin deficiency) can lead to onset of COPD at a young age.^279,280 Smoking or other irritants can induce persistent and recurrent inflammation of the bronchi and alveoli, which progressively damages the airways and lung parenchyma, accelerates the physiological decline of lung function with age, and ultimately results in airflow limitation and lung damage.^281,282 COPD encompasses several subtypes, including emphysema, chronic bronchitis, and small airway disease.²⁸³ The symptoms of chronic bronchitis are cough and sputum production, which is caused by inflammation in the central airway epithelium and mucus-producing glands, usually related to an innate immune response to inhaled irritants. This pathological inflammation may result in mucus hypersecretion, reduced mucociliary clearance, and increased permeability of the airspace epithelial barrier.²⁸⁴ Emphysema is caused by the enlargement of the distal airspaces beyond the terminal bronchioles, resulting from the destruction of airway walls. Emphysematous lung destruction may reduce maximal expiratory airflow.²⁸⁵ Small airway disease refers to obstruction in the smaller conducting airways, typically less than 2 mm in diameter. Inflammation and peribronchial fibrosis can lead to fixed airway obstruction of the small airways and destruction of the alveolar attachments in the outer wall of the small airways.²⁸³ COPD currently has no cure. Smoking cessation is the only proved cost-effective intervention to reduce the risk of COPD onset and progression.²⁸⁶ Pharmacological treatment mainly aims to improve airflow and ameliorate symptoms and exacerbations. Bronchodilators, such as selective β2-adrenergic agonists, anticholinergics, and theophylline, are used to relieve symptoms in COPD patients. Glucocorticoids are recommended for severe COPD or for individuals experiencing frequent exacerbations.²⁸⁷ However, effective treatment to reduce the progressive decline in lung function in COPD is still lacking.

4.1.2. Epidemiological studies of exercise intervention in COPD

The positive effects of physical activity and exercise for patients with COPD are well documented.288, 289, 290 Generally, COPD patients exhibit sedentary behavior and lower physical activity levels due to respiratory symptoms. Physical inactivity in COPD is associated with poor health outcomes, such as dyspnea, skeletal muscle dysfunction, exercise intolerance, and decreased health-related quality of life.²⁹¹ Therefore, improving physical activity levels in COPD is crucial.

Most patients with COPD, even the more severe cases, can benefit from physical activity or exercise interventions. Physical activity has been verified to improve COPD prognosis, delay COPD development, and decrease hospitalization and mortality rate.²⁸⁸ A population-based cohort study revealed that smokers who engage in moderate to high levels of regular physical activity experience a slower decline in lung function and have a lower risk of developing COPD.²⁹² Additionally, physical activity has positive physiological, social, and psychological effects and is linked to a variety of health outcomes among individuals with COPD. Unsupervised home-based physical activity interventions, such as daily walking, can alleviate fatigue and muscle weakness, enhance health-related quality of life, and improve emotional well-being.²⁹³

The protective effects of exercise against COPD are related to improved cardiovascular function,²⁹⁴ skeletal muscle oxidative capacity, and fatigue resistance.²⁹⁵ Aerobic exercise training alleviates inflammation in both the plasma and broncho-alveolar lavage fluid (BALF) in individuals with COPD, and it decreases emphysema-like changes in the alveoli as well.²⁹⁶ Exercise-induced improvements are observed after 6–8 weeks of exercise, and longer programs tend to yield more pronounced benefits.²⁹¹

Both moderate continuous training and HIIT for 10 weeks improved systolic cardiac function and aerobic capacity in COPD patients.²⁹⁴ High-intensity exercise training in severe COPD patients improved their exercise time in cardiopulmonary exercise testing, resulting in reduced ventilatory requirements and exertional dyspnea.²⁹⁷ Compared to the improvements in exercise capacity and quality of life that are achieved through aerobic training alone, additional strength training is associated with a significantly greater increase in muscle strength and mass in patients with COPD.²⁹⁸ For older patients with COPD who are unable to accomplish land-based exercise, water-based physical exercise training enhances their exercise capacity and quality of life.²⁹⁹

4.1.3. Basic research on the effect of exercise in COPD

The protective effects of physical exercise on COPD have been demonstrated in animal models as well. Exercise training is found to alleviate the development of smoking-induced COPD in rodents,³⁰⁰ an effect comparable to the benefits of exercise in COPD patients.²⁹² Studies on COPD animal disease models have revealed that exercise not only improves pulmonary conditions but also addresses metabolic comorbidities associated with COPD. Two months of running can improve pulmonary function, ameliorate emphysema,³⁰¹ reduce airway remodeling,³⁰² and rescue glucose metabolic dysregulation and insulin resistance³⁰³ in COPD mice.

The protective effect of exercise against lung injury in COPD is mediated by immune system regulation, such as anti-inflammatory mediators and antioxidant enzymes related to physical exercise.³⁰⁴ Exercise attenuates the recruitment of immune cells, including neutrophils and macrophages in the BALF of mice exposed to smoking.³⁰⁴ Furthermore, exercise substantially suppresses ROS levels in BALF and attenuates 8-isoprostane expression in lung tissues of COPD mice. The reductions in IL-10, tissue inhibitor of metalloproteinases 1 (TIMP1), and CuZn superoxide dismutase are rescued by moderate-intensity exercise. This training also increases glutathione peroxidase-expressing cells, thus alleviating pulmonary inflammation and disease progression.³⁰⁰ Additionally, aerobic exercise has been shown to decrease other inflammatory cytokines or pro-fibrotic molecules, such as C-X-C motif chemokine ligand 1 (CXCL1), IL-1β, IL-17, TNF-α, matrix metalloproteinase 9 (MMP9), transforming growth factor-β (TGF-β), and sirtuin 1 (SIRT1) in lung tissues of COPD mice.³⁰⁵

Studies on COPD animal models have demonstrated that the anti-inflammatory effects of exercise are related to the wingless-type mmtv integration site family (Wnt)/β-catenin/peroxisome proliferator-activated receptor gamma (PPARγ)³⁰⁶ and signal transducer and activator of transcription 3 (STAT3)³⁰⁷ signaling pathways. Furthermore, it has been reported that irisin, which is secreted from the muscle during exercise, may exert protective effects against oxidative stress via activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), thereby leading to amelioration of emphysema in COPD.³⁰¹ Another study found that 2 months of treadmill running attenuated diaphragm atrophy in COPD mice, which was related to the inhibition of the Ras homolog gene family, member A (RhoA)/Rho-associated coiled-coil containing protein kinase (ROCK) signaling pathway.³⁰⁸

4.1.4. Animal exercise intervention studies and models for COPD

COPD disease models are typically induced in mice and rats by exposure to cigarette smoke, intra-tracheal lipopolysaccharide (LPS), or intranasal elastase stimulations.³⁰⁹ Treadmill running and swimming are commonly applied to investigate the effects of exercise on COPD animal models.^{296,302,306,310} Among these, aerobic exercise is predominantly performed through treadmill running, especially moderate-intensity running in rodent models.^302,304,306 The training protocol generally involves running at a speed corresponding to 50%–60% VO_2max for 30–60 min per day, with intervention durations ranging from 8 weeks to 12 weeks.^{301,305,308,296,311} Moreover, HIIT on the treadmill has also been applied in COPD-related studies, comprising 10 intervals of 4 min at 80%–90% VO_2max and 2–4 min of recovery at 50%–65% VO_2max for 4–6 weeks.^312,313 For swimming-based exercise interventions, rodents undergo training sessions for 60 min/day over a period of 8 weeks.³¹⁴ The key parameters of treadmill running and swimming as forms of exercise training in COPD animal models are summarized in the following content (Supplementary Table 5).

4.1.4.1. Treadmill running

Treadmill running is the most commonly used mode for investigating the effects of exercise on COPD. In these studies, male C57BL/6J, ICR, or A/JOlaHSD mice at the age of 4–8 weeks, and Wistar rats at the age of 8–10 weeks are frequently used.^{303,306,311,315} A pre-training before formal exercise protocols is crucial to acclimate the animals to the treadmill environment. This pre-training is conducted at a low-intensity (from 3.3–5.0 m/min of running for mice,^300,305,306 8–10 m/min for rats³¹⁶) for 3–7 days. For example, studies have applied pre-training for 15 min on the treadmill at a constant speed of 3.3 m/min (0.2 km/h) at a 25% incline for 3 days in mice³⁰⁰ or, in rats, an adaptation exercise for 5 min at 8–10 m/min at 0° incline for 5 days.³¹⁶ There is no specific requirement for the incline of the treadmill; both a flat slope³⁰¹ or 25%³⁰⁴ incline have been utilized in these experiments.

A maximal exercise capacity test is conducted after the adaptation phase to determine the maximum aerobic capacity, which can be performed as follows: begin with a 5-min warm-up at 5 m/min, followed by gradual increases in running speed (e.g., 2 m/min every 3 min) until the mice become exhausted. The running speed at the time of exhaustion is recorded as the maximum exercise speed.³⁰⁶ Due to the progression of disease, it is recommended to perform the maximal exercise capacity test every 3–4 weeks throughout the experiment period (e.g., at Weeks 0, 4, and 8 during an 8 week-running program).³⁰⁶ It should be noted that although exercise intensity should ideally be determined according to VO_2max (or VO_2peak), some studies defined the exercise intensity groups according to the percentage of maximal running speed. This alternative approach is adopted because VO_2max measurement requires a specific metabolic chamber, which is not always easily obtainable.³⁰⁶

Low-intensity: There are limited studies exploring the role of low-intensity exercise on COPD. The protective effect of low-intensity treadmill running against COPD can be observed after 4 weeks of intervention.³⁰⁵ For example, after a pre-training phase conducted at a speed of 3.3 m/min for 20 min/day for 3 days, a formal exercise training was initiated at 11.8 m/min for 60 min/day for 4 weeks, which was found to effectively attenuate LPS-induced COPD in mice.³⁰⁵ To improve pulmonary function and inflammation in cigarette smoke-induced COPD, running on the treadmill at 35% of maximal running speed for 60 min/day for 8 weeks was found to alleviate BALF inflammation and reduce lung emphysema, although its effectiveness was lower compared to moderate-speed exercise (55% of maximal running speed) in mice.³⁰⁶

Moderate-intensity: Studies on the effect of exercise on COPD animal models mostly use treadmill running at moderate-intensity. The speed of treadmill running is set at 50%–60% VO_2max (some studies used 50%–55% of maximal exercise speed, although this is not recommended).^{296,300,301,311} Exercise sessions can last 30–60 min per day, 5–7 days/week for 8–12 weeks.^301,304,308 The beneficial effect of moderate-intensity treadmill running became significant after 8 weeks of exercise³⁰⁸ and was even more evident after 12 weeks of exercise, specifically related to the enhanced anti-inflammatory and anti-oxidative capacity of the lung.^300,301,304

High-intensity: HIIT can improve COPD as well. This protocol is typically conducted by alternating between 4 min of high-intensity running (80%–90% VO_2max or VO_2peak) and 2–4 min of moderate-intensity running (50%–65% VO_2max or VO_2peak) with a 0°–25° incline for 60 min/day, 5 days/week for 4–6 weeks.^312,313,315 For instance, a 6-week HIIT program consisting of 10 intervals (4 min of running at 90% VO_2max, followed by 2 min of recovery at 60% VO_2max) performed 5 days/week on a 25° incline treadmill improved exercise capacity and left ventricular systolic function in COPD mice.³¹³ Due to respiratory dysfunction, continuous high-intensity training is seldom used in studies investigating exercise interventions for COPD. One study has reported that running on a treadmill at 85% of maximal exercise speed for 60 min/day, 6 days/week for 8 weeks can alleviate lung structural changes, improve pulmonary function, and reduce inflammation in COPD mice.³⁰⁶ It should be noted that this study categorized different exercise groups according to the percentage of maximal exercise speed instead of VO_2max, which is not recommended in animal exercise studies. Nonetheless, this study reported that the effects of high-speed running (85% of maximal exercise speed) on alleviating pulmonary inflammation and improving lung function were less compared to moderate-speed running (55% of maximal exercise speed).³⁰⁶

4.1.4.2. Swimming

Studies exploring the effect of swimming exercise on COPD are very limited. In a study using the waterpipe tobacco smoking exposure-induced COPD rat model, 60 min of swimming (with 2%–5% BW load by caudal dumbbells) for 8 weeks effectively prevented the induction of pro-inflammatory cytokines.³¹⁴ Prior to formal swimming training, an adaptation is necessary. The specific procedures for adaptation vary across different studies. The general principle is to acclimate animals to the swimming environment by gradually increasing the duration of swimming within 4–6 days. For example, a pre-training is conducted as follows: Day 1, 30 s of swimming, twice a day (2 h interval); Day 2, 2 min of swimming, twice a day (2 h interval); Day 3, 10 min of swimming, three times a day (5 min intervals); and Day 4, 15 min of swimming, twice a day (5 min interval).³¹⁰ Following this adaptation phase, mice underwent a formal exercise program by swimming 30 min/day for a total of 10 days, which can decrease the inflammatory cells in BALF, reduce pro-inflammatory cytokines, and induce IL-10 in the lung tissues of mice exposed to diesel exhaust particles.³¹⁰

4.1.4.3. Intervention time

The timing of exercise interventions should be decided according to the study’s objectives. To investigate the effect of exercise on COPD pathogenesis, interventions are typically implemented concurrently with risk exposure.^300,301,305 To examine the therapeutic or rehabilitative potential of exercise on COPD, it is essential to establish a disease model before commencing exercise training in COPD animal models.^303,308

4.1.5. Evaluation metrics in COPD research with an exercise intervention

To evaluate the effect of exercise in COPD experimental animal studies, it is recommended to analyze parameters related to respiratory function, histopathological characteristics, pulmonary inflammation, and oxidative stress (Table 5). Respiratory function is measured by a pulmonary function test system to evaluate respiratory flow, pressure, and volume wave.^{300,303,304,306,308} Histopathological characteristics, including the degree of lung emphysema, fibrosis, and pulmonary remodeling, can be detected by hematoxylin–eosin staining, Masson’s trichrome staining, and Sirius Red staining.^{300,301,303,305,306,308} Due to the impact of inflammation on the onset and progression of COPD, the levels of inflammatory response and oxidative stress are considered important indicators. These related markers include the number of immune cells (such as macrophages, neutrophils, lymphocytes) in BALF and serum as well as the expression levels of inflammatory-related cytokines (IL-1β, IL-10, IL-17, TNF-α, TGF-β, etc.) and antioxidative molecules in the disease models.^300,303, 304, 305, 306

Table 5.

Evaluation metrics in COPD research that could be examined with an exercise intervention.

Detection index	Parameters	Reference
Pulmonary respiratory function	Pulmonary ventilation function (e.g., MV, FEV, PEF), lung volume (e.g., FVC, FRC), airway resistance, and pulmonary compliance (e.g., Cydn) are measured by a Pulmonary Function Test System	300,303,304,306,308
Histopathological examinations	Lung emphysema: airspace size and rupture of alveolar septa by H&E staining Pulmonary fibrosis: Masson’s trichome staining, Sirius Red staining	300,301,303,305,306,308
Inflammation response	Immune cell numbers (e.g., neutrophils, macrophages, lymphocytes, eosinophils) in BALF and serum Levels of cytokines (e.g., IL-1β, IL-10, IL-17, TNF-α) in BALF and lung tissues	300,303, 304, 305, 306
Oxidative stress	The levels of SODs, GPx, GSH, MDA, MPO ROS production	300,305

Abbreviations: BALF = broncho-alveolar lavage fluid; COPD = chronic obstructive pulmonary diseases; Cydn = dynamic lung compliance; FEV = forced expiratory volume; FRC = functional residual capacity; FVC = forced vital capacity; GPx = glutathione peroxidase; GSH = glutathione; H&E = hematoxylin-eosin; IL = interleukin; MDA = malondialdehyde; MPO = myeloperoxidase; MV = minute ventilation; PEF = peak expiratory flow; ROS = reactive oxygen species; SOD = superoxide dismutase; TNF-α = Tumor necrosis factor-α.

4.1.6. Recommendations

• •To explore the effects of exercise on COPD, treadmill running at both moderate-intensity and HIIT is generally implemented, while swimming exercise is rarely used in the COPD animal studies.
• •To effectively alleviate inflammatory stress and improve pulmonary function in COPD animal models, treadmill running at moderate-intensity (50%–60% VO_2max; approximately 12.5–18.0 m/min in mice^300,301,308 and 13.5–17.5 m/min in rats^296,311) for 30–60 min per day for a duration of 8–12 weeks is recommended.
• •In terms of exercise duration, the effect of moderate-intensity treadmill running became significant after 8 weeks of exercise³⁰⁸ and was even more evident after 12 weeks of exercise, specifically related to the enhanced anti-inflammatory and anti-oxidative capacity of the lung.^300,301,304
• •Compared to low- and high-intensity continuous running exercise, moderate-intensity treadmill running is more efficient to reduce pulmonary inflammation and improve pulmonary function in COPD animal models.³⁰⁶
• •Continuous high-intensity training is seldom used in studies investigating exercise interventions for COPD. HIIT-conducted by alternating between 4 min of high-intensity running (80%–90% VO_2max or VO_2peak) and 2–4 min of moderate-intensity running (50%–65% VO_2max or VO_2peak) with a 0°–25° incline for 60 min/day, 5 days/week for 4–6 weeks—is effective for improving COPD.^312,313,315

5. Animal exercise intervention studies for AD

5.1. AD

5.1.1. Introduction to AD

The WHO reported that 55 million people globally were living with dementia in 2021, and this number is expected to reach 139 million by 2050. AD is the most common type of dementia, accounting for 60%–80% of cases.³¹⁷ In the USA, 6.9 million adults aged 65 or older are living with Alzheimer’s dementia today; this number will rise to 13.8 million by 2060. Medicare payments for individuals with dementia are triple those without the condition.³¹⁸ The rapidly aging population worldwide will lead to a continuous increase in AD cases. This trend imposes substantial economic and psychological burdens on patients, their families, and caregivers. Additionally, it exerts significant pressure on healthcare systems and broader socioeconomic structures.

AD is a neurodegenerative disorder. Its primary clinical manifestations include memory decline and cognitive dysfunction. The pathogenesis of AD involves multiple complex mechanisms. Genetic factors account for 60%–80% of AD risk, with over 40 genetic risk loci identified. The apolipoprotein E (ApoE) allele shows the strongest genetic correlation with AD development.³¹⁹ The disease pathology begins in the cellular phase with accumulation of beta-amyloid (Aβ) and triggers the spread of Tau pathology.³¹⁹ Specifically, Aβ abnormally aggregates in the brain to form senile plaques, which are considered core pathogenic factors of AD, causing neuronal dysfunction and death.³²⁰ Concurrently, Tau protein undergoes abnormal phosphorylation, forming neurofibrillary tangles that disrupt microtubule structures and impair intracellular transport and signaling.^321,322 The loss of cholinergic neurons (choline acetyltransferase positive (ChAT⁺) cells) impairs neural signal transmission and exacerbates cognitive function.³²³ Additionally, activated microglia mediate inflammatory responses and oxidative stress, leading to neuronal damage.³²⁴ Collectively, these pathological processes interact and promote the occurrence and progression of AD.

5.1.2. Epidemiological studies of exercise intervention in AD

Exercise interventions play a vital role in preventing and managing AD. A recent comprehensive systematic review and meta-analysis of 29 prospective cohort studies demonstrates that higher levels of physical activity are significantly associated with reduced risk of AD.³²⁵ Different exercise intensities show varying effects on cognitive function. Low-intensity exercises that focus on stretching, balance, range of motion, and relaxation, as well as moderate-intensity activities such as brisk walking, are beneficial. However, only HIIT has shown significant enhancements in cognitive function, with effects lasting up to 5 years.³²⁶ Both aerobic and resistance training have been demonstrated to improve cognitive performance, memory, and overall brain volume in older adults.³²⁷ For instance, a 5-kg increase in grip strength is associated with a reduced overall dementia and AD risk ratio, indicating that resistance training can effectively prevent AD.³²⁸ Moreover, moderate-to-high-intensity weekend physical activity decreases the risk of dementia, stroke, Parkinson’s disease, and mental health issues.³²⁹ It is important to note that animal studies provide valuable mechanistic insights into the neuroprotective effects of exercise, which may help us better integrate exercise interventions into the management of AD.^330,331

5.1.3. Basic research on the effect of exercise in AD

Basic research reveals multiple beneficial mechanisms of exercise in AD. Exercise promotes the degradation and clearance of Aβ, thereby reducing its accumulation in the brain.^331,332 Meanwhile, exercise regulates the activity of related kinases, such as glycogen synthase kinase-3 beta (GSK3β), which decreases the abnormal phosphorylation of Tau protein and inhibits the formation of neurofibrillary tangles.333, 334, 335 Additionally, exercise promotes neurogenesis through elevating the levels of exercise-induced metabolic factors (e.g., ketone bodies, lactate) and muscle-derived myokines (e.g., cathepsin-B, irisin), which stimulate the production of neurotrophins such as BDNF.335, 336, 337 Furthermore, exercise exhibits anti-inflammatory and antioxidant effects and reduces levels of inflammatory factors (IL-1β, IL-6, IL-8, TNF-α, TGF-β, monocyte chemotactic protein-1 (MCP-1), and NLRP3) and oxidative stress, thereby protecting neurons from damage and ameliorating the pathophysiological hallmarks of AD (e.g., Aβ deposition).^334,336 In summary, exercise plays a crucial role in improving AD through multiple mechanisms: reducing Aβ accumulation, lowering Tau protein phosphorylation, promoting neurogenesis, and exerting anti-inflammatory and antioxidant effects.^336,337

5.1.4. Animal exercise intervention studies and models for AD

Various exercise types such as treadmill running, swimming, and wheel running have been utilized to assess their protective roles against cognitive dysfunction and neurodegeneration in AD models. These investigations have used diverse AD models, including amyloid precursor protein/presenilin-1 (APP/PS1) and other transgenic mice (predominantly C57BL/6 background), intracerebroventricular infusion of Aβ oligomers in Swiss mice, and STZ-induced sporadic rat AD models. To ensure comprehensive evaluation, these studies involve both female and male subjects, with the typical age range being 3.0–16.0 months for mice and 2.5 months for rats.333, 334, 335^,338, 339, 340, 341, 342 Understanding the pathophysiological characteristics of these animal models is crucial for designing appropriate exercise protocols and accurately interpreting the results.

A significant characteristic of genetically modified AD mice is the early manifestation of aging-like symptoms compared to WT counterparts. This distinctive feature can influence their exercise capacity, allowing them to achieve the VO_2max at relatively lower treadmill running speeds.^334,343,344 This adaptation needs to be taken into consideration for exercise protocol design and intensity classification. For example, when 3-month-old female APP/PS1 transgenic mice were initially subjected to treadmill running, they began at a speed of 5 m/min for 5 min, which was then increased to 8 m/min for another 5 min, and finally elevated to a maximum speed of 11 m/min for the remaining 20 min; this speed represents 45%–55% of their VO_2max.³⁴⁴ Meanwhile, starting at a speed of 5 m/min for 5 min, then increasing to 12 m/min for another 5 min, and finally reaching a maximum speed of 15 m/min for 20 min represents moderate-intensity exercise at 60%–70% VO_2max.³⁴⁴ In another AD model, 16-month-old female and male transgenic mice expressing human tau23 under the control of the neuron-specific enolase (NSE) promoter (Tg-NSE/htau23 mice) performed treadmill running exercise at 12 m/min or 19 m/min for 60 min/day, producing VO_2max levels of 50%–60% (moderate-intensity) and 70%–80% (high-intensity), respectively.³³⁴ Thus, in AD animal models with exercise intervention, both genetic background and age can influence the correspondence of running speed to VO_2max.

Treadmill running protocols typically involve sessions of run speeds at low/moderate intensity (5–15 m/min), high-intensity (19 m/min), or HIIT consisting of 10 cycles—each comprising 4 min at high intensity (20 m/min) and 2 min at low intensity (10 m/min). These sessions are conducted 4–5 days/week for 3–5 months, resulting in significant cognitive improvements and reduced Aβ accumulation.^333,334,338, 339, 340, 341 Swimming exercises are typically performed in female and male transgenic mice (C57BL/6 background, 13.0–16.0 months), Swiss mice (2.5–3.0 months), or SD rats (2.5 months). The protocol involves 20–60 min/day for 3–4 weeks, leading to enhanced cognition and reduced neuroinflammation.^345,346 Wheel running exercise also involves genetically modified AD mice (C57BL/6J background, 6–8 weeks or 3–5 months old, both female and male). The mice are either housed in cages with running wheels for voluntary exercise over 4–9 months or trained on running wheels at 18 rounds/min for 40 min/day, 6 days/week over a period of 5 months, which leads to increased BDNF levels and enhanced spatial learning and memory.^{331,335,347,348} It is noteworthy that although many AD studies utilize both female and male mice, the experimental data do not specifically address sex-based differences in exercise responses.^334,349 In some studies that reported results separately by sex, they generally failed to demonstrate significant sex-specific variations in exercise efficacy.³³¹ The neuroprotective effects of estrogen have been reported in neurological disease research. Thus, the inclusion of both sexes helps to establish the generalizability of exercise-induced neuroprotection against AD pathology rather than to investigate sex-specific mechanisms. The following sections will delve into the key aspects related to treadmill running, swimming, and wheel running exercises in AD research, highlighting the specific protocols and outcomes associated with each type (Supplementary Table 6).

5.1.4.1. Treadmill running

Treadmill running exercise is extensively utilized in AD research as a therapeutic intervention. Like other studies, when using treadmills to observe the effect of exercise, mice and rats require pre-training to help them quickly adapt to the equipment. Generally, in the AD mice models, pre-training lasts between 2 and 6 days, with training speeds ranging from 5 to 12 m/min. The pre-training speed correlates with subsequent exercise intensity. For instance, when the maximum training speed is 11 m/min, mice undergo pre-training at speeds of 5–8 m/min.³³⁹ Whereas for a maximum training speed of 19 m/min, AD mice are pre-trained at 12 m/min.³³⁴ Pre-training for AD rats can be more complex, requiring 2 weeks (3 days/week) of training: in the first week, running at 4  m/min for 15 min, then 6 m/min for 30 min, and 8 m/min for 45 min; in the second week, 8 m/min for 45 min, 12 m/min for 45 min, and finally 18 m/min for 45 min.³⁴²

After finishing the pre-training, exercise interventions at varying intensities are administered to AD mouse and rat models. In mice, low/moderate-intensity exercise sessions involve running at speeds ranging from 5 to 15 m/min, whereas high-intensity sessions may reach speeds up to 20 m/min. These interventions are conducted over extended periods of 3–5 months.^334,339, 340, 341 For AD rat models, speeds of up to 18 m/min are used for durations of 8 months.³⁴² Evidence suggests that in 16-month-old Tg-NSE/htau23 mice, high-intensity treadmill exercise at 19 m/min may offer superior protective effects compared to moderate-intensity exercise at 12 m/min.³³⁴ Specifically, these exercise regimens require a longer duration (3–8 months) to effectively reduce Aβ accumulation, decrease p-Tau, enhance synaptic plasticity, and improve cognitive function in AD mouse and rat models.^{333,334,338,341,342}

Low/moderate-intensity: Low/moderate-intensity training in AD mouse models uses speeds of 5–15 m/min for 30–60 min/day, 4–5 days/week, over a duration of 3–5 months.^333,334,338, 339, 340, 341 In AD rat models, exercise is conducted at speeds of 18 m/min for 45 min/day, 3 days/week, over 8 months.³⁴² Additionally, low/moderate-intensity aerobic exercise with a variable range of speeds is commonly employed in AD research. For example, in 3-month-old APP/PS1 mice, the pre-training protocol consists of 15 min/day for 6 days: the speed is 5 m/min on the first 2 days, which increases to 8 m/min on Days 3–4 and reaches 12 m/min on Days 5–6.³⁴¹ The formal treadmill exercise protocol involves 5 m/min for 5 min, 8 m/min for 5 min, 12 m/min for 30 min, and 5 m/min for 5 min (45 min/day in total), 5 days/week for 12 weeks.³⁴¹ This exercise regimen reduced Aβ deposition in the hippocampus of APP/PS1 mice, possibly by regulating a disintegrin and metalloproteinase 10 (ADAM10) and beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) levels and decreasing cholesterol-mediated lipid raft formation.³⁴¹

High-intensity: High-intensity training or HIIT protocols are also employed in studies exploring exercise interventions for AD. In these exercises, the maximum running speed of AD mice reaches 19–20 m/min, with sessions lasting 60 min/day for 4–5 days/week over 3–5 months.^334,338 Specifically, in 3-month-old APP/PS1 mice, each training session consists of 10 cycles (each cycle comprising 4 min at high intensity (20 m/min) and 2 min at low intensity (10 m/min), therefore 60 min/day in total) performed 4 days/week for 20 weeks. This HIIT effectively reduced memory deficits and Aβ deposition in the mice.³³⁸

5.1.4.2. Swimming

Swimming exercise is frequently employed in research examining the preventive and therapeutic effect of exercise on AD.^345,346,350 Compared to other forms of aerobic exercise, swimming offers a low-impact, full-body workout that may offer unique neuroprotective benefits. The experimental setup involves plastic barrels (60 cm depth × 45 cm diameter) filled with water, where mice swim in groups of four. The training protocol begins with 2 days of 10 min of pre-training, followed by regular sessions of 20 min/day for 3 weeks (C57BL/6 mice) or 60 min/day for 5 weeks (Swiss mice), 5 days/week.³⁴⁵ It is important to note that different mouse strains have varying exercise tolerances. Swiss mice undergo a progressive adaptation protocol: initial 10-min sessions for 2 days to minimize water-induced stress, followed by gradual duration increases until reaching 60 min (typically achieved by Day 5), with final sessions of 60 min/day, 5 days/week for 5 weeks; and C57BL/6 background mice follow a protocol of 20 min/day for 3 weeks.³⁴⁵ For STZ-induced sporadic AD rat models (2.5 months old), their training starts at 10 min/day, increasing by 10 min every 2 days until reaching 1 h/day, which is maintained through Day 26.³⁴⁶ Notably, swimming can produce beneficial neuroprotective effects against AD in a shorter exercise duration (3–5 weeks) compared to other modalities. These exercise regimens have been shown to reduce neuroinflammation, decrease oxidative stress, and enhance neuronal survival.^345,346

5.1.4.3. Wheel running

Wheel running represents another important exercise type in AD research.^{331,335,347,348} This exercise type uses wheels of 12-cm diameter and requires 4–9 months to achieve significant benefits in C57BL/6 background AD models. Studies typically utilize mice aged 6–8 weeks or 3–5 months.^{331,335,347,348} In these studies, young mice (6–8 weeks) demonstrate average running distances of 3.5–4.0 km/day, monitored hourly by revolution counter.³⁴⁷ While wheel running experiments are often used for voluntary exercise training, some AD studies have employed wheel running for involuntary exercise. For example, in 5-month-old male APP/PS1 mice, wheel running exercise was conducted at 18 rounds/min for 40 min/day, 6 days/week for 5 months. This exercise protocol effectively increased lysosomal function, reduced memory defects, and decreased Aβ deposition.³⁴⁸ Both voluntary and involuntary wheel running protocols demonstrate therapeutic potential in AD research.

5.1.4.4. Intervention time

Unlike other disease models with predictable pathological timelines, AD studies require unique considerations for intervention timing due to the heterogeneous nature of transgenic mouse/rat models. Exercise intervention timepoints are typically defined by major pathological milestones, such as before or after amyloid plaque deposition (which varies among different mouse strains) or before or after the onset of behavioral/cognitive deficits (with strain-specific and symptom-specific temporal variations).^351,352 Consequently, exercise interventions in AD research span extensive time periods, with some studies initiating regimens prior to significant pathological onset and continuing through disease progression,³³¹ while others begin after established symptoms.^333,334 The selection of intervention timing should be determined based on specific research objectives and the pathological characteristics of the chosen animal model. Importantly, various forms of exercise consistently demonstrate beneficial effects in slowing AD pathological progression when maintained for adequate duration, regardless of when intervention is initiated, highlighting the therapeutic flexibility and robust neuroprotective potential of exercise across different disease stages.

5.1.5. Evaluation metrics in AD research with an exercise intervention

Exercise demonstrates significant potential as a non-pharmacological intervention for AD. To assess the effects of exercise in AD animal models, researchers typically employ a series of behavioral tests and biomarkers for comprehensive evaluation (Table 6). Behavioral assessments, such as the Morris water maze, Y-maze test, novel object recognition test, and tail suspension test, are utilized to evaluate learning ability, memory, and emotional functions in mice.^{333,334,339,340,345} Biochemical analyses focus on measuring key pathological markers of AD, including Aβ accumulation and Tau protein hyperphosphorylation, to assess the impact of exercise on disease progression.^{333,334,338,341} Additionally, oxidative stress markers, such as antioxidant enzymes like SOD and catalase (CAT), are evaluated to understand the antioxidative effects of exercise interventions.³⁵³ Neuroinflammatory cytokines, including IL-1β, IL-6, TNF-α, and NLRP3, are measured to assess the anti-inflammatory benefits of exercise.^324,335,347 Histological and immunohistochemical analyses of neuronal survival, synaptic plasticity, and neurogenesis involve markers such as BDNF, synaptophysin, postsynaptic density protein-95.^331,345,347,354, 355, 356 Collectively, the integration of behavioral tests, biochemical assays of pathological proteins, oxidative stress, and inflammatory markers, along with histological evaluations, provides a comprehensive framework for assessing the effectiveness of exercise intervention in AD.

Table 6.

Evaluation metrics in Alzheimer’s disease research that could be examined with an exercise intervention.

Detection index	Parameters	Reference
Behavior function  Neurological function scale  Memory function  Motor function  Emotion	A DNMP task to measure pattern separation Eight-arm radial arm maze, Y-maze, Morris water maze, contextual fear conditioning, novel object recognition, Barnes maze test Rotarod test, grip strength test, gait analysis Tail suspension test, elevated plus maze, open field test, social anxiety behavior test	331,333, 334, 335, 336,338,345,347
Cell morphology and function analysis	Golgi staining, RV-CAG-GFP, Electrophysiological analysis (e.g., LTP), CBF determination by LSCI	331,336,345,347
Motion-induced factor detection	BDNF, PSD-95, SYP, IL-6, and FNDC5, TGF-β	331,354, 355, 356
Key pathological markers	Aβ/Tau protein and plaques, activity of Neprilysin and insulysin (or insulin-degrading enzyme)	324,331, 332, 333, 334,338,345,347
Inflammatory response	Activated glia (GFAP+, Iba-1+ cell), IL-1β, IL-6, IL-8, TNF-α, TGF-β, MCP1, Toll-like receptors (TLR1, TLR2, TLR4 and TLR6), CD68, and NLRP3	324,335,347
Oxidative stress	SOD activity measurement, CAT activity measurement, OGG1 staining	334,338
Cell death	BrdU and EdU labeling, ChAT+ cell immunofluorescence (cholinergic neurons)	335,347

Abbreviations: Aβ = beta-amyloid; BDNF = brain derived neurotrophic factor; BrdU = thymidine analog 5′-bromo-2′-deoxyuridine; CAT = catalase; CBF = cerebral blood flow; CD68 = cluster of differentiation 68; ChAT = choline acetyltransferase; DNMP = delayed nonmatching to place; EdU = 5-ethynyl-2′-deoxyuridine; FNDC5 = fibronectin type III domain-containing protein 5; GFAP = glial fibrillary acidic protein; Iba-1 = ionized calcium binding adaptor molecule-1; IL = interleukin; LSCI = laser speckle contrast imaging; LTP = long-term potentiation; MCP1 = monocyte chemoattractant protein-1; NLRP3 = nucleotide-binding oligomerization domain (NOD)-, leucine-rich repeat (LRR)- and pyrin domain-containing protein 3; OGG1 = 8-Oxoguanine DNA Glycosylase 1; PSD-95 = postsynaptic density protein-95; RV-CAG-GFP = retroviral green fluorescent protein reporter; SOD = superoxide dismutase; SYP = synaptophysin; TGF-β = transforming growth factor-β; TLR = toll-like receptors; TNF-α = tumor necrosis factor-α.

5.1.6. Recommendations

• •Aerobic exercises including treadmill, swimming, and wheel running effectively promote the degradation of Aβ and Tau protein and plaques.331, 332, 333, 334^,345
• •Relative to other diseases, AD research utilizing treadmill exercise commonly employs variable-speed protocols within specific intensity ranges (e.g., progressive protocols such as 5 m/min for 5 min, 8 m/min for 5 min, then 12 m/min for 30 min) to optimize neuroplasticity outcomes over extended intervention periods of 3–5 months.^333,342
• •Swimming demonstrates beneficial neuroprotective effects against AD with shorter intervention durations (3–5 weeks)^345,346 compared to other exercise modalities such as treadmill running (3–5 months) or wheel running (4–9 months), making it an efficient exercise option for AD research protocols.
• •When implementing swimming protocols in AD animal models, the selection of appropriate exercise sessions must take consideration into the animal’s physical capabilities and strain-specific tolerances. For example, Swiss mice can swim for 60 min/day, while C57BL/6 mice are typically limited to 20 min/day, and SD rats can swim for 60 min/day.^345,350 These duration differences reflect inherent strain-specific exercise capacities and stress tolerance levels that must be considered during protocol design.
• •Regarding exercise intervention timing: In AD models, pathological symptoms do not occur at strictly defined time points but rather within broad temporal windows that vary significantly among different transgenic mouse/rat strains. Exercise intervention timing should be strategically selected based on specific research objectives and the pathological characteristics of the chosen animal model.
• •VO_2max is a key indicator for assessing aerobic exercise intensity; however, the detection and control of VO_2max are rarely mentioned in AD research.
• •The lasting protective effects of exercise warrant further investigation, as human studies demonstrate retention of hippocampal improvements for at least 5 years following a 6-month exercise regimen.³²⁶ Animal studies reveal that mice maintained elevated muscle succinate dehydrogenase activity for 2 weeks after 4 weeks of voluntary wheel running, with levels returning to baseline after 4 weeks of cessation.³⁵⁷ While research on long-term exercise effects in AD models remains limited, 8 months of treadmill training (2–10 months of age) followed by 8 months of detraining still demonstrated sustained cognitive benefits when assessed at 18 months of age, indicating prolonged neuroprotective effects.³⁴² These findings highlight the need for systematic investigation of exercise protocols that maximize long-term therapeutic benefits in AD research.

6. Other factors to note in animal exercise intervention studies for chronic diseases

6.1. Sex and age of animals

In addition to the exercise training facilities, environment, and protocols, the animal’s own factors can affect the effect of exercise. The sex differences of animal physical activity have been previously reviewed.³⁵⁸ The results of existing studies on sex differences of exercise have shown that female rodents are more active than male ones. Early studies reported that ovariectomy significantly decreased wheel-running activity in female rats.³⁵⁹ Although the specific mechanism is not yet clear, there is evidence suggesting that the estrogen receptor alpha (ERα) and the dopamine pathway may be involved in mediating this phenomenon.³⁵⁸ ERα, which regulates mitochondrial function and metabolic homeostasis in females, is expressed in both skeletal muscle and the heart.³⁶⁰ Interestingly, the deficiency of ERα expression in the heart may not only lead to cardiac dysfunction in females but also induce obesity in female mice via heart-derived extracellular vesicles.³⁶¹ This effect is observed in female mice but not in male mice. The different ERα expression is also likely to impact the response to exercise. Additionally, the cardiac physiological hypertrophic responses are more evident in female mice.³⁶² In a traumatic peripheral nerve injury model, low-intensity continuous treadmill running is only effective in male mice, while high-intensity interval treadmill running is only effective in female mice to enhance axon regeneration.³⁶³ In a genetically modified AD mouse model, 4 months of treadmill exercise more effectively improved spatial learning and memory abilities and delayed changes in the white matter of female mice with early AD than in male mice.³⁶⁴

Age is a critical determinant in rodent exercise studies, representing a significant methodological consideration in experimental design. While chronic diseases predominantly affect aging populations, most preclinical exercise intervention studies have been conducted in young rodents, which highlights a notable limitation in current research. This age-related discrepancy requires careful attention in experimental protocols and results interpretation. Age-associated decline in exercise capacity is well-documented in rodent models. Spontaneous wheel running activity is significantly reduced in rats, especially in rats after 7–8 months of age.^365,366 The treadmill running speed should be differently set according to the reduced VO_2max in old rodents compared to young- or middle-aged rodents. For example, compared to running at 36 m/min on a 15% slope for 1 h in young rats, old rats run at a progressively increased speed until 15 m/min by the fourth week, and then receive an increment of 5% slope until the final workload of 15% slope.³⁶⁷ For old rats, swimming with a 2% overload for only 20 min per day can exert anti-oxidant beneficial effects in the myocardium.³⁶⁸

6.2. Genetic background of animals

Interindividual differences in exercise capacity have been increasingly reported in both animal experimental studies and in humans, which is at least in part related to genetic background.^369,370 Exercise capacity and blood pressure responses during treadmill exercise tests are differentially associated with α-adrenergic receptors, endothelial NO synthase, and bradykinin B2 receptor in men and in women.³⁷⁰ Similarly, mice with endothelial NO synthase or β3-adrenergic receptor deficiency had significantly reduced exercise capacity during voluntary wheel running.¹²⁶ Endurance exercise capacity is linked to an increased activity of citrate synthase (CS), the key enzyme of the tricarboxylic acid cycle.^371,372 In addition to the run time, speed, and distance, measurement of CS activity in the tibialis anterior and gastrocnemius can also be a critical indicator for exercise capacity in animals.^27,295,373 In mice after 4 weeks of voluntary wheel running, the CS activity (µmol/mg protein/min) was increased by about 35% in the tibialis anterior and gastrocnemius muscles in non-transgenic C57BL/6 mice, whereas the myosin heavy chain null mice had distinct patterns of muscle adaptation to the wheel running.²⁹⁵ In mice, after 4 weeks of swimming training, the CS activity (µmol/mg protein/min) was also increased in the mixed gastrocnemius muscles but to a lesser extent, and no difference was found between the forkhead box protein O1 (FoxO1) KO mice and WT mice.²⁷

6.3. Comparison of different exercise models for disease interventions

Numerous studies have shown that exercise can prevent the development of chronic diseases. However, in addition to selecting appropriate exercise modalities (e.g., treadmill running, voluntary wheel running, or swimming), it is also important to compare the effect of different exercise modes (intensity, frequency, or duration) in physiological and pathological models. During the process of natural aging, moderate-intensity exercise training for 24 weeks was shown to improve cardiac function and modulate cardiac structural and metabolic changes, while HIIT did not.³⁷⁴ Some studies have shown that both aerobic exercise training and HIIT can improve cardiac function after MI; however, HIIT (with an incline) may cause structural damage of myocardial mitochondria.¹³² In an obesity-related heart failure with preserved ejection fraction rat model, 8 weeks of moderate-intensity continuous training improved stroke volume, end-diastolic volume, and cellular arrhythmogenicity, while HIIT did not.³⁷⁵ In addition, a single bout of treadmill running for 20 min and a short-term treadmill running program of 20 min per day for 2 weeks differentially regulated histone post-translational modifications in rat hippocampus.³⁷⁶ More recently, it was shown that although low-intensity exercise improved cardiac health, moderate-intensity (12 m/min, 5° incline) and high-intensity (18 m/min, 5° incline) treadmill running exercise exacerbated high-calorie-diet-induced cardiac dysfunction through a redistribution of circulating lipid to the heart.²⁰³ Therefore, exercise is not always protective against disease although it is natural for mice and rats to run. Different exercise regimens can essentially influence the functional effects of exercise, which may be closely related to distinct cellular and molecular change patterns.

6.4. Translation of animal exercise studies to human disease interventions

Basic research of animal exercise intervention studies in the prevention and treatment of chronic diseases is essential to deepen our understanding of the mechanisms of the beneficial effects of exercise.^9,377 Meanwhile, researchers should also consider the translational significance of their animal exercise intervention studies for potential clinical applications to treat diseases. Firstly, one frequent question is whether and how the exercise models used in animal experiments can be applied to the prevention and treatment of diseases in humans. Although exercise intensity can be classified according to VO_2max both in humans and animals, the transition from animal experiments to humans requires great caution. However, the exercise models used in animal experiments will provide positive support and guiding significance for exercise rehabilitations in clinical treatment. Secondly, whether clinical patients can tolerate an exercise training program during their disease progression is also a question worth considering. Indeed, both preventive and therapeutic exercises deserve to be studied in animal exercise experiments.¹⁵ Well-designed cohort studies and randomized controlled trials are greatly needed to reveal the effects of exercise on the prevention and treatment of chronic diseases. Thirdly, exercise mimetics is, so far, a cutting-edge concept that needs to be further investigated and applied to the treatment of diseases, especially for patients who are not suited for exercise training during the progression of their disease. Accumulating evidence has reported the functional role of beneficial molecules that are regulated by exercise in the treatment of chronic diseases, including but not limited to signaling proteins,^373,378 transcription factors,^{27,142,379,380} non-coding RNAs,^28,381,382 other RNA modulations,³⁸³ and secreted factors.³⁸⁴ Some exercise-regulated molecules have also been identified in exercised healthy people or patients, indicating their potential to be used as biomarkers for disease diagnosis or prognostic assessment.³⁸⁵ Based on the identified exercise-responsive molecules, the combined application of gene therapy, small molecule inhibitors, metabolites, etc. will provide new strategies for disease interventions.^386,387

7. Conclusion

This expert consensus provides recommendations on fundamental research of animal exercise intervention studies for chronic diseases including stroke, CAD, COPD, obesity, T2DM, and AD. Based on expert discussions, the consensus provides specific recommendations for animal exercise intervention studies in terms of exercise models (frequency, intensity, time, type, etc.), model constructions, and physiological effects of exercise (functional, structural, biochemical, and disease-specific) in such disease models. The sex, age, and genetic background of animals, comparisons of different exercise models, and translational significance of animal exercise intervention studies have been stated in this consensus. We anticipate that this consensus provides practical recommendations for the improved design and conduct of fundamental research in animal exercise intervention studies. In turn, this will promote our understanding of the effects of exercise in chronic disease interventions and further develop physical exercise or exercise-mimic interventions for disease prevention and treatment.

Authors’ contributions

JX, MX, and YQ designed the concept and structure of the position paper and provided substantial revisions to the draft manuscript; YB and DZ designed the concept and structure of the position paper. All authors were actively involved in writing and composing subsections of the position paper. All authors have read and approved the final version of the manuscript, and agree with the order of presentation of the authors.

Declaration of competing interests

The authors declare that they have no competing interests. Given their both roles as board members, JX and AR had no involvement in the peer review of this article and had no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to another journal editor.