Effect of astaxanthin supplementation on cycling performance, muscle damage biomarkers and oxidative stress in young adults: a randomized controlled trial

Abstract

Background

The consumption of dietary supplements to enhance endurance performance and fitness is gaining popularity among professional athletes and nonathletes. Astaxanthin (AST), a natural ketocarotenoid, has been tested for its ergogenic, antioxidant, and tissue protective properties in young male adults.

Methods

Ten physically active male adults (22.5 ± 0.9 years) were randomized into placebo or AST trials (according to CONSORT), and consumed placebo or AST (28 mg/d) supplements orally for 4 days. On day-4, participants performed an exhaustive cycling challenge at 75% maximum rate of O₂ uptake (V̇O₂max), and the time to exhaustion (TTE) was recorded. Blood and gaseous samples were collected before, during, and immediately after cycling to determine changes in muscle damage, inflammation, oxidative stress, and substrate utilization.

Results

Short-term AST supplementation significantly enhanced exercise performance, as we found longer TTE in the AST trial (85.41 ± 4.42 min) than in the placebo trial (72.11 ± 2.24 min). Statistical analysis revealed a significant larger effect (P < 0.001; partial eta squared (η²p) = 0.71) on enhanced TTE with AST. Exhaustive exercise-induced muscle damage, indicated by increased creatine kinase (CK) and lactate dehydrogenase release, was significantly (P < 0.05) decreased by AST. A significant time and treatment interaction effect for CK (P = 0.039, η²p = 0.217) indicating potential attenuation of muscle damage by AST. In addition, lipid peroxidation, as evidenced by increased malondialdehyde levels during and immediately after exercise, was substantially inhibited by AST (P < 0.05). However, inflammatory markers (tumor necrosis factor-alpha and C-reactive protein) did not respond to either AST supplementation or exercise challenge. Substrate utilization during and after exercise appeared to be similar in both trials. Importantly, AST supplementation had no adverse effects on the ‘profile of mood states’ among participants.

Conclusions

Short-term AST supplementation could be a nutritional ergogenic aid to enhance endurance performance and attenuate exhaustive exercise-induced muscle damage or oxidative stress in young adults.

Trial registration

The study was approved by the Human Research Ethics Committee of China Medical University Hospital (CMUH111-REC3-081) and registered at clinicaltrials.gov under registration number NCT06593535 (dated 05-09-2024).

Supplementary Information

The online version contains supplementary material available at 10.1186/s13102-025-01221-3.

Introduction

Moderate-intensity exercise training, either aerobic or resistance, has been associated with enhanced physical fitness and well-being in humans through positive physiological adaptations [1]. Reactive oxygen species (ROS) that are produced during exercise serves as intracellular messengers to regulate physiological adaptation [2]. In contrast, excessive production of ROS during high-intensity exercise involves in the propagation of oxidative stress, inflammation, and tissue damage, which results in poor athletic performance [2–4]. High-intensity training sessions or overtraining can induce adverse effects through increased skeletal muscle damage, muscle soreness and fatigue [5]. This was evidenced by elevated biomarkers of muscle damage (creatine kinase (CK), lactate dehydrogenase (LDH)), lipid peroxidation (malondialdehyde (MDA)) and inflammation after high-intensity exercise [6, 7]. In this state, participants may discontinue their exercise challenge due to energy deficits or fatigue, which limits maximum performance in competitions. Therefore, attenuation of muscle damage, oxidative stress or inflammation through dietary supplements might be a practical strategy to boost the exercise performance in athletes. Previous studies have emphasized the importance of the total antioxidant capacity of an individual to counteract the exhaustive exercise-induced muscle damage and metabolic fluctuations [8]. Nutraceutical supplements possess antioxidant properties are widely used in sports nutrition, and research on their ergogenic properties is gaining popularity [9, 10]. Recent human studies used the supplements like quercetin, green tea extract, and conjugated linoleic acid, and emphasized their beneficial effects on endurance performance, oxidative stress, muscle damage or substrate utilization during exercise challenge [11–14].

Astaxanthin (AST) is a naturally occurring lipid soluble red‒orange carotenoid that is primarily found in marine species, fish, crustacea, microalgae and some birds [15–17]. AST is composed of two β-ionone ring systems that are linked by a polyene chain and contain oxygenated keto and hydroxyl moieties, which are responsible for its powerful antioxidant activity [16–18]. Intake of AST has shown numerous health benefits in humans, including improved antioxidant and inflammatory systems under different stress conditions [17, 19]. Randomized controlled trials (RCTs) have reported that AST supplementation for 90-day (4 mg/day) attenuated CK release and ROS production, while promoted antioxidant status in young soccer players against soccer training and soccer exercise [19, 20]. In contrast, recent RCTs showed inefficiency of 28-day AST treatment, either 8 mg/day [21] or 12 mg/day [22], in decreasing the exercise-induced muscle damage, muscle soreness or inflammation in trained adults. However, AST supplementation for 28-day (4 mg/day) [23] or 7-day (12 mg/day) [24], reportedly increased the cycling performance; whilst the fat oxidation rate increased only with short-term (7-day) [24]. Contrary, a relatively higher dose of AST (20 mg/day) for 28-day had no effect on exercise performance or substrate utilization in trained cyclists [25]. AST intake for 4-week (12 mg/day) also showed no effect on fat oxidation, but decreased carbohydrate oxidation rates during cycling exercise in individuals with overweight [26]. Taken together, these findings indicate that the ergogenic or physiological benefits of AST are equivocal, in terms of substrate oxidation rates or time trial/performance, which might be due to the variance in dose and/or duration of supplementation.

The total dose of AST used in these previous trials ranged from 84 to 560 mg, while the duration of the trials ranged from 7- to 90-day, and findings on the recommendation of AST as a nutraceutical ergogenic aid are inconclusive. The studied daily doses of AST ranging from 4 to 20 mg appears to be well-tolerated with no significant side effects [15, 19–21, 23, 25]. However, the lack of precision on the optimal dosage or treatment duration, particularly in sports science limiting its adoption as an ergogenic or nutraceutical supplement. Either short-term daily doses of AST up to 100 mg or long-term daily doses between 8 and 12 mg reported to be safe, but higher doses may cause increased frequency in bowel movement and red-colored stool [27–29]. Choi et al. administered a normal-dose (5 mg) and high-dose (20 mg) of AST for 3-week, and observed no adverse effects or toxicity in overweight or obese adults, instead, improved antioxidant capacity, decreased lipid peroxidation and alleviated subjective symptoms like fatigue were reported [27]. AST intake for 8-week (12 mg/day) reported to be beneficial on overall mood, especially on decreasing the negative mood state variables, depression and fatigue in adults [30]. In this context, short-term consumption of AST at a safe dose might be warranted for athletes to maximize their performance or recover from exercise-induced fatigue or stress. It is worth to note that no study investigated the short-term (4-day time-frame) effect of AST at a daily dose of 28 mg on cycling performance, physiological changes or mood profile in adults. Therefore, we designed this study to examine the effect of short-term safe-dose AST supplementation on exercise performance among young college adults. We hypothesized that short-term AST supplementation may mitigate the exhaustive exercise-induced muscle damage and/or physiological anomalies in adults.

Materials and methods

Participants

Ten healthy, physically active male college students from the National Taichung University of Education were recruited and completed the study (between June and September 2022). The mean age of the participants was 22.5 ± 0.9 years, and the mean body mass index (BMI) was 22.9 ± 0.83 kg/m². During the experimental period, participants were instructed to avoid any form of intensive exercise, intake of caffeinated or alcoholic beverages, or intake of other nutritional supplements that could influence antioxidant or inflammatory response. All participants maintained their regular healthy lifestyle, including adherence to eating habits and sleeping patterns, which was monitored through daily logs. Participants were provided with a normal diet consisting of 60% carbohydrates, 25% fat, and 15% protein. The study protocols were clearly explained to each participant, and the written informed consent was obtained. The entire study design and methodology were reviewed and approved by the Human Research Ethics Committee of China Medical University Hospital (CMUH111-REC3-081, Taichung, Taiwan) and registered at clinicaltrials.gov under registration number NCT06593535 (dated 05-09-2024). This study was conducted in accordance with the ethical standards of the committee responsible for human experimentation, and with the Helsinki Declaration of 1975 (revised 2013). Power analysis was conducted using G*Power (version 3.1.9.4) on the observed means and standard deviations of the time to exhaustion performance in AST (85.41 ± 4.42 min) and placebo (72.11 ± 2.24 min) trials (n = 10). The calculated effect size (Cohen’s d) was 3.47, which indicates a large effect. Given this effect size, the sample size provides sufficient statistical power (> 0.95), confirming the robustness of the findings.

Study design and experimental procedures

In this randomized crossover-designed study (single-blind), participants were assigned to AST or placebo trials. Randomization was performed using the GraphPad QuickCalcs Website (https://www.graphpad.com/quickcalcs/randomize1/) in five blocks with stratification into either AST or placebo groups. Upon completion of a 4-day intervention with placebo or AST supplements, participants were crossed over for alternative AST or placebo supplements, which was completed in 4-day. Between the crossover trials, participants had a one-week washout period to nullify the effects of the supplements (Fig. 1). Given the actual shorter duration of AST intake (4-day) and its plasma half-life time of approximately 15 h [31], one-week washout period was deemed sufficient. Besides, one-week washout period was well adopted in previous human studies, which supplemented AST for 4-week at the dose of 6 or 12 mg/day [22, 32]. Participant inclusion and randomization was performed in accordance with the Consolidated Standards of Reporting Trials (CONSORT) statement. Further stepwise details were shown in Fig. 1, and the CONSORT checklist was provided as Supplementary Table 1.

Fig. 1.

CONSORT flow chart. This figure shows the flow of participants through the trial according to the criteria recommended in the CONSORT Guidelines

For the preliminary test, the maximum oxygen consumption (VO₂max) of the participants was determined seven days prior to the study. Participants in the placebo or AST trials received their respective supplements for three continuous days after breakfast, and the 4th supplement was received on the day of the experiment. Participants rested for one hour following low-calorie breakfast (300 calories) and supplementation, and then performed an exhaustive cycling exercise at 75% VO₂max on a stationary ergometer bike (Monark 894E, Varberg, Sweden). This single bout of cycling exercise challenge consisted of a 5-min warm-up (50-watt) and continued until exhaustion at 75% VO₂max, where the individual maximum ‘time to exhaustion (TTE)’ was recorded [12]. Blood and gaseous samples were collected five times—one hour before (B), at the beginning (0-min), during (20-min, 40-min), and immediately (E)—after exhaustive cycling exercise challenge. The detailed experimental design and sampling procedure were depicted in Fig. 2. Soon after the exercise challenge, the participants were asked to complete the profile of mood state (POMS) questionnaire.

Fig. 2.

Experimental design and stepwise protocols

Astaxanthin and placebo supplementations

During the supplementation period (4-day), participants reached the laboratory at 7 AM and consumed their low-calorie breakfast. The breakfast composed of 60% carbohydrates, 25% fat, and 15% protein in a total of 300 calories, and this was same for both trials. Following breakfast, either AST (AstaReal^®, Japan, Fig. 3A) or placebo capsules were orally supplemented. Participants in the AST trial consumed 28 mg of AST per day in the form of capsules. Each capsule contained 4 mg of AST, which means 7 capsules per day for four days, and the total dose was 112 mg. The AST capsules were obtained from the Fuji Chemical Industries Co. (Toyama, Japan), and the required numbers of capsules were consumed directly, without mixing or combining with other ingredients. The total dose of AST in our study (112 mg) is comparable to the dose of a previous human study by Earnest and colleagues [23], who claimed the beneficial effects of AST over a period of 28-day. Based on previous dose safety studies, 28 mg of AST per day is within the safe and tolerable range for humans [27–29, 31], and we assumed shortest duration (4-day) could have similar beneficial effects like 28-day [21–23]. A dose of AST up to 40 mg/day for a period of 4-week is considered to be safe and tolerable [28, 29].

Fig. 3.

The molecular structure of astaxanthin (A). Average time to exhaustion with 75% V̇O₂max cycling challenge in placebo- and astaxanthin (AST)-supplemented trials (B). The values are expressed as the means ± SE (n = 10). The results are significantly (**) different between the placebo and AST trials at P < 0.001

Determination of VO₂max and time to exhaustion

Participants performed a VO₂max test on a stationary ergometer bike (Monark 894E, Varberg, Sweden) while wearing a gas exchange facemask connected to the analyzer (Cortex Biophysik, Leipzig, Germany). Initially, the intensity was set at a workload of 0.5 kg for 4 min and then gradually increased by 0.5 kg every 2 min at each subsequent stage. The rotational speed was set at 60 revolutions per minute (RPM) throughout the test until the person reached exhaustion. Individual VO₂max was considered valid if 2 of the following 3 conditions were met: (1) the respiratory exchange ratio (RER) was greater than 1.10, (2) the change in relative VO₂max was < 2 ml/kg/min and reached a steady state, and (3) the heart rate (HR) reached the predicted maximum (208 − 0.7 × age) [33, 34]. The value obtained from the Y-axis is the oxygen uptake (ml/kg/min), and the value obtained from the X-axis is the oxygen uptake corresponding to the workload intensity (weight of the weights). The value of VO₂max (100%) was determined when the value on the Y-axis reached a plateau. Then, the value of VO₂max was multiplied by 0.75 to obtain the 75% VO₂max of the person. According to a previous study, a 75% maximal oxygen uptake load was used as the cycling time to exhaustion or TTE [12].

Assessment of the RER, fat oxidation rate and carbohydrate oxidation rate

The pulmonary oxygen consumption (VO₂) and carbon dioxide production (VCO₂) during exercise were used to determine metabolic substrate utilization. The gaseous exchange ratio during exercise reflects the relative contributions of carbohydrates and fat to energy metabolism. The RER was calculated by dividing the VCO₂ value by the VO₂ value ().

Fat oxidation and carbohydrate oxidation rates in response to cycling challenge were determined using the VO₂ and VCO₂ data. Based on previous studies [35], the fat oxidation rate was calculated using the following equation:

Similarly, the carbohydrate oxidation rate was determined by the following equation as described in the Péronnet and Massicotte study [35].

Evaluation of muscle damage biomarkers

Blood samples were collected at five different time points and centrifuged at 1000 × g for 15 min. The resulting serum was stored at -20 °C and used for biochemical analyses. Muscle damage biomarkers, including creatine kinase (CK), lactate dehydrogenase (LDH), and uric acid (UA), were measured in the serum. The CK (EC2.7.3.2), LDH (EC 1.1.1.27), and UA (C97792) concentrations were estimated using commercial analytical reagents (Beckman Coulter). An automated clinical chemistry analyzer was used to measure CK and LDH levels on a Beckman Coulter AU5800 (Beckman Coulter Inc., CA, USA).

Evaluation of total antioxidant capacity and lipid peroxidation

The antioxidant homeostasis in terms of Trolox equivalent antioxidant capacity (TEAC) was measured using Cano et al. (2000) method [36]. Serum samples were diluted 50-fold and mixed with peroxidase, ABTS⁺ (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid), hydrogen peroxide, and water. The mixture was incubated for 60-min to produce stable ABTS⁺ cationic radicals. Then, the diluted serum and ABTS⁺ cationic radicals were added to 96-well plates and allowed to react for 10-min. The absorbance values were read at 734 nm using a microplate reader (Versa Max, Molecular Devices, USA). The scavenging ability of antioxidants (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Trolox) was determined using a curve, and the amount of Trolox equivalent to the test serum’s inhibition rate was calculated. Next, the level of malondialdehyde (MDA), a standard biomarker of lipid peroxidation, was determined by an ELISA kit provided by the Caman Company (Cayman Chemical Company, Michigan, USA). As described in the protocol, the reaction mixture was boiled at 90–100 °C for 60-min, and the absorbance at 550 nm was measured using an ELISA plate reader (Tecan GENios, A-5082, Austria). The values are expressed as micromoles of MDA per liter.

Determination of the inflammatory response

Changes in inflammatory biomarkers, including tumor necrosis factor-α (TNF-α) and high-sensitivity C- reactive protein (hs-CRP), were detected in the serum of both trials. The proinflammatory cytokine TNF-α was analyzed with a commercial ELISA kit (BioLegend, San Diego, CA) according to the manufacturer’s instructions. The absorbance was read at 450 nm within 15 min by an enzyme immunoassay (Tecan GENios, A-5082, Austria). Concentrations of C-reactive protein (CRP) were measured using a human ELISA kit (E-80CRP, Immunology Consultants Laboratory, Inc., Newberg, OR, USA). The sample absorbance was read at 450 nm within 30-min.

Assessment of the profile of mood state (POMS) of participants

The profile of mood states (POMS) was initially developed by McNair and colleagues [37], and is a noninvasive and convenient method for evaluating the mood states of athletes in sports. Later McNair et al. [38] published the POMS-brief version questionnaire comprising only 30 adjectives grouped in the same six dimensions, which was even more convenient to adopt. Therefore, the POMS-brief version was chosen for this study to evaluate the mood states of participants. After the TTE challenge, participants completed the questionnaire, and their responses were analyzed using t-test analysis. The results were shown in six dimensions, one positive: (1) vigor; and five negatives: (2) tension, (3) depression, (4) anger, (5) fatigue and (6) confusion.

Statistical analyses

The experimental data were analyzed with IBM SPSS version 22.0 for Windows (IBM Crop., Armonk, NY, USA). All data are presented as the means ± standard errors (SEs). The paired t-tests were conducted to analyze the statistical significance between trials for ‘time to exhaustion’ and also for variables in POMS. For the blood biomarkers, a two-way ANOVA (visit × time) with repeated measures was applied to determine the statistical significance. Significance levels were set at α = 0.05 for all analyses. The effect sizes were estimated using partial eta squared (η2p), and interpreted according to the Cohen’s standard. The threshold of effect size was categorized and interpreted as small (d = 0.20/η2 = 0.01), medium (d = 0.50/η2 = 0.06), and large (d = 0.80/η2 = 0.14) with their respective values [39].

Results

No participant was dropped during the intervention either in placebo or in AST trial. The intervention with ten participants was successfully executed in compliances with the CONSORT guidelines, and all assessments were done as planned.

AST promotes cycling performance in young college adults

The ergogenic properties of AST were determined by recording the maximum ‘time to exhaustion’ of cycling performance at 75% VO₂max. We found that cycling performance in terms of TTE was significantly (P = 0.001) longer with AST supplementation (85.41 ± 4.42 min) than with placebo (72.11 ± 2.24 min). Most participants in the AST trial exhibited longer TTE than those in the placebo trial (Fig. 3B), which suggests that short-term AST supplementation enhances exercise performance in young adults. According to the statistical analysis, there is a significant and larger effect size with treatment (Cohen’s ‘d’=1.42; η²p = 0.071), indicating AST accounted for a greater improvement in TTE. The increased cycling time with AST might be attributed to its protective role against muscle damage or oxidative stress induced by exhaustive exercise challenge.

AST reduces muscle damage during exhaustive cycling performance

The possible protective effects of AST against cycling challenge-induced muscle damage were examined by monitoring the muscle damage biomarkers (CK and LDH). Exhaustive cycling-induced muscle damage in the placebo trial was represented by elevated CK concentrations. However, this CK elevation was not observed in the AST trial. To be specific, CK concentrations in AST trial were significantly lower (P < 0.023) during cycling (20-min) and immediately after cycling compared with that of in placebo, at respective time points (Fig. 4A). We further noticed a significant time and treatment interaction (P = 0.039, η²p = 0.217) with medium effect size (Supplementary Table 1). Similarly, elevated LDH concentrations immediately after cycling were observed in the placebo trial. However, AST supplementation showed significantly reduced LDH release (P < 0.004) immediately after exercise (Fig. 4B). The time and treatment interaction effects were non-significant for the LDH (η²p = 0.035) as shown in Supplementary Table 2. These results imply that short-term AST supplementation might be effective in reducing the exhaustive cycling-induced muscle damage in young adults.

Fig. 4.

Changes in creatine kinase (CK) (A) and lactate dehydrogenase (LDH) concentrations (B) before and during a single bout of exhaustive cycling challenge in the AST (-•-) and placebo (-○-) trials. AST: astaxanthin, B: before exercise, E: immediately after exercise. The values are expressed as the means ± SE (n = 10). For statistical significance (P < 0.05), * indicates difference between the trials at respective time points, and + indicates difference compared to before exercise

AST inhibits lipid peroxidation without altering the inflammatory response

Changes in inflammatory biomarkers, such as TNF-α and hs-CRP, were measured before, during, and immediately after cycling. As shown in Fig. 5A, the circulating TNF-α concentration did not significantly differ between the placebo and AST trial at any time point. Similarly, changes in hs-CRP concentrations were also not significantly different between the trials after exhaustive exercise (Fig. 5B). Both TNF-α and hs-CRP showed no interaction effect with time and treatment, but the effect size appears to be large (η²p = 0.459) for hs-CRP (Supplementary Table 2).

Fig. 5.

Changes in serum tumor necrosis factor-α (TNF-α) (A), high-sensitivity CRP (hs-CRP) (B), total antioxidant capacity (TEAC) (C), and malondialdehyde (MDA) concentrations (D) before and during a single bout of exhaustive cycling challenge in the AST (-•-) and placebo (-○-) trials. AST: astaxanthin, B: before exercise, E: immediately after exercise. The values are expressed as the means ± SE (n = 10). For statistical significance (P < 0.05), * indicates difference between the trials at respective time points. and + indicates difference compared to before exercise

We noticed that antioxidant capacity remained stable with AST supplementation as well as with a single bout of cycling challenge (Fig. 5C). Interestingly, cycling caused lipid peroxidation in the placebo trial, as evidenced by significantly increased MDA levels during (40-min) and immediately after exhaustive cycling (P < 0.012). However, AST supplementation attenuated cycling-induced lipid peroxidation, as we noticed significantly decreased MDA levels (P < 0.021) during exercise (40-min) and immediately after exercise (Fig. 5D). The time and treatment interaction effects were non-significant for both antioxidant capacity and lipid peroxidation with the given dosage of AST (Supplementary Table 2). Nevertheless, the decreased MDA levels suggest that AST pre-supplementation could avoid lipid peroxidation against exhaustive cycling without altering the inflammatory system in healthy adults.

Effect of AST supplementation on substrate utilization

The effect of short-term AST supplementation on substrate utilization during exercise challenge is presented in Fig. 6. Our data revealed that the RER, fat oxidation and carbohydrate oxidation rates significantly increased (P < 0.001) during cycling (20-min and 40-min), but these trends were similar in both the AST and placebo trials. The increased RER and carbohydrate oxidation rate with exercise was peaked at 20-min of cycling and tended to decrease thereafter in both trials (Fig. 6A & C). In contrast, the increased fat oxidation rate was peaked at 40-min of cycling, and reached baseline at the exhaustion point (Fig. 6B). However, the time and treatment interaction effects were non-significant for RER (η²p=0.004), fat oxidation (η²p = 0.008) and carbohydrate oxidation rates (η²p = 0.023) (Supplementary Table 3). Short-term oral AST supplementation appears to be ineffective in influencing the substrate utilization during exhaustive cycling challenge.

Fig. 6.

Respiratory exchange ratio (RER) (A), fat oxidation rate (B), and carbohydrate oxidation rate (C) before and during a single bout of exhaustive cycling challenge in the AST (-•-) and placebo (-○-) trials. AST: astaxanthin, B: before exercise, E: immediately after exercise. The values are expressed as the means ± SE (n = 10). For statistical significance (P < 0.05), + indicates difference compared to before exercise

AST shows no adverse effects on mood profile after exhaustive exercise

The POMS scores assessed soon after exhaustive exercise in both trials are presented in Table 1. The average scores of each subscale of the POMS, including tension, depression, anger, vigor, fatigue, and confusion, were not significantly different between the placebo and AST trials. The scores of four negative domains (tension, depression, fatigue, and confusion) were slightly lower, and the score of one positive domain (vigor) was slightly higher in the AST trial, however not statistically significant. A paired t-test analysis also showed no clinically significant changes in mood states after exhaustive exercise (Supplementary Table 4). Collectively, these findings revealed that short-term AST supplementation does not influence the mood state of participants, as assessed by the POMS.

Table 1.

Profile of mood States (POMS) after a single bout of time to exhaustion cycling challenge in Astaxanthin (AST) and placebo treatments. Values are expressed as mean ± SE (n = 10)

POMS Scores	Placebo	AST	P values
Tension	2.56 ± 0.85	2.2 ± 0.64	0.74
Depression	1.67 ± 0.55	1.56 ± 0.38	0.87
Anger	1.67 ± 0.58	2.67 ± 0.75	0.11
Vigor	10.44 ± 1.46	11.37 ± 1.88	0.48
Fatigue	6.89 ± 1.45	5.44 ± 1.64	0.20
Confusion	4.00 ± 1.01	3.89 ± 1.12	0.91

Discussion

To the best of our knowledge, this is the first human study to demonstrate the short-term effect of AST supplementation (28 mg/day for 4-day) on cycling performance, physiological responses and mood states in healthy young men. We found that the given dosage of AST notably enhanced the cycling performance (TTE) compared to placebo, suggesting the ergogenic benefits. AST supplementation significantly decreased exhaustive cycling-induced muscle damage possibly through decreased CK (during and immediately after cycling) and LDH (immediately after cycling) activities. This protective property of AST was accompanied by a significant inhibition of lipid peroxidation (MDA) against exhaustive cycling-induced elevation. Nonetheless, the given dose of AST for 4-day did not influence total antioxidant capacity, inflammatory response or substrate utilization during exercise challenge. However, it is important to note that AST exhibited no adverse effects on the mood swings of participants assessed by POMS immediately after cycling challenge. Taken together, short-term AST supplementation exhibited ergogenic effects, which may be attributed to its protective role against cycling challenge-induced muscle damage or lipid peroxidation in healthy young adults.

One of the important findings in our study was that short-term AST supplementation promoted endurance performance in young adults. During intense exercise or competition, exhaustion occurs when muscles are unable to perform contractile activities due to a lack of sufficient ATP supply. This situation could be due to the lack of fuel sources from carbohydrate, fat or glycogen metabolism or dysfunction of the ATP-generating system by intracellular ROS [ 40, 41 ]. Strategies such as short-term resting between the exercise sessions or pre-supplementation of nutraceutical compounds with antioxidant property is practically beneficial for restoring energy levels and improving exercise performance. The dietary supplement industry, particularly the sales of sports nutrition supplements to enhance exercise performance, is growing globally, and this market is projected to reach US$ 27 billion by 2027 [ 42 ]. In fact, evidence-based optimization of dose and duration of such supplements is highly important. Establishing scientifically validated dose and consumption duration of supplements in real practice is essential to maximize the performance and minimize the incidence of undesired complications or adverse effects [ 10 ].

In this study, we provided convincing evidence that short-term oral AST supplementation for 4 days (28 mg/day) improved maximum cycling performance in physically active young adults. A human study on professional cyclists reported that AST supplementation for 28 days (4 mg/day) improved exercise performance [23]. A recent study showed that AST supplementation for 7 days (12 mg/day) also improved the time to complete a 40-km cycling trial in trained male cyclists [24]. In contrast, supplementation with higher doses of AST (20 mg/day) for 4 weeks does not improve either endurance performance or fat oxidation in well-trained male cyclists [25]. AST supplementation for 30 days (12 mg/day) also showed no effect on aerobic capacity, however improved exercise recovery in healthy adults [43]. The prolonged time to exhaustion and higher muscle glycogen levels in AST-fed mice (4 weeks) explaining the potential glycogen-sparing effect of AST [44]. Previous studies suggested that AST could improve exercise performance and recovery due to its potent antioxidant properties [15, 23, 32, 44]. However, the effects of AST on exercise performance, recovery and skeletal muscle glycogen restoration during exercise are equivocal in humans. The beneficial effects of AST on exercise performance appear to be associated with its dose and duration of intake. However, our findings suggest that even short-term AST supplementation can enhance endurance performance, possibly by reducing muscle damage markers and oxidative stress in healthy young adults.

Although moderate-intensity exercise is beneficial, exhaustive exercise can induce muscle damage, inflammation, impair antioxidant homeostasis, and subjective discomfort in humans [2, 45]. The magnitude of exhaustive exercise-induced muscle damage in our study was evidenced by a notable elevation of circulating CK and LDH levels, while inflammatory markers (TNF-α and hs-CRP) remained unchanged during cycling. However, AST pretreatment appears to attenuate this damage by inhibiting CK and LDH concentrations, and this protective effect corresponding with enhanced exercise performance. A preclinical study showed that AST supplementation for 7 days (100 mg/kg/day) exerted impressive protective effects in a rat model of multi-organ injury, by reversing the tissue injury biomarkers, LDH, creatinine and CK-MB, and improving the survival [46]. A previous human study on young soccer players revealed that 90 days AST supplementation (4 mg/day) attenuated muscle damage, as indicated by a significant decrease in both CK and LDH activities [19]. Furthermore, 4-week AST supplementation (12 mg/day) mitigated delayed-onset muscle soreness (DOMS) and soreness without affecting resistance training performance in trained males following exercise-induced muscle damage (EIMD) [47]. These beneficial effects of AST might be associated with its natural antioxidant and tissue protective properties. In contrast, a recent study by Waldman et al. (2023) showed that 4-week AST supplementation (12 mg/day) did not influence the biomarkers of muscle damage, DOMS or inflammation in resistance-trained male adults following EIMD protocol [22]. Another RCT demonstrated that AST supplementation for 4-week (8 mg/day) had no counteractive effect on exhaustive 2.25 h running-induced muscle damage (CK) or muscle soreness in male or female adults [21]. These disparities in the ability of AST to attenuate muscle damage markers are possibly due to the differences in AST dosage, training status of participants or training protocol that adopted to induce muscle damage.

In agreement with previous studies, we found elevated MDA levels following exhaustive exercise challenge in placebo trial, indicating increased lipid peroxidation due to increased ROS production during intense exercise [2, 45]. These highly unstable ROS eventually react with biomolecules, including lipids, especially polyunsaturated fatty acids (PUFAs), and trigger lipid peroxidation [45]. Excessive lipid peroxidation exacerbates muscle tissue damage, and thereby impairs athletic performance [48]. Therefore, attenuation of lipid peroxidation or oxidative stress by dietary antioxidant supplements during exercise is an effective strategy to enhance and/or maintain endurance performance. To emphasize the protective effect of AST, we measured the lipid peroxidation and antioxidant status following exhaustive exercise challenge. We demonstrated that AST supplementation inhibited exhaustive cycling-induced MDA elevation, which is similar to the reduction of muscle damage markers. The decreased MDA levels in exercised AST trial indicating its efficiency in suppression of exercise-induced lipid peroxidation. The suppression of lipid peroxidation with AST may be attributed to its unique molecular properties as a lipid-soluble carotenoid. AST structure enables its ability to permeate through the phospholipid bilayer of mitochondria and scavenges intracellular ROS [16]. Goto et al. suggested that the conjugated polyene moiety of AST can trap radicals in the phospholipid membrane, while the terminal ring moieties of AST can trap radicals both at the membrane surface and in the membrane, which could inhibit lipid peroxidation [49]. An animal study showed that AST supplementation for 45 days (1 mg/kg bodyweight) increased muscle antioxidant status, controlled oxidative stress and delayed exhaustion [50]. In a human study, intake of AST for 4 weeks (6 mg/day) increased glutathione content without affecting the substrate utilization/fat oxidation during exercise [32]. In our study, the total antioxidant capacity remained unchanged before and after exercise in AST trial. We assume that such a stable antioxidant status may have contributed to scavenge ROS, and thereby mitigated lipid peroxidation/muscle damage and improved performance in the AST trial.

Next, we examined the effect of AST supplementation on substrate utilization during a 75% VO₂max cycling challenge in young adults. We found that the RER, fat oxidation and carbohydrate oxidation rates increased at 20- and 40-min during cycling in both the placebo and AST trials. Our findings suggest that short-term AST supplementation (28 mg/day, 4-day) might be insufficient for maximizing the substrate utilization during exercise. Similar to our findings, AST supplementation for 28-day (4 mg/day), which total dose is equal to our study, had no effect on fat or carbohydrate oxidation in trained cyclists, despite the improved endurance performance [23]. Another RCT showed that 4-week AST supplementation at the dose of 12 mg/day decreased carbohydrate oxidation rate, but had no effect on fat oxidation rate during graded cycling test in individuals with overweight [26]. Furthermore, a relatively high dose of AST (20 mg/day for 4-week) did not increase either the whole-body fat oxidation rate during exercise or endurance performance in trained cyclists [25]. In contrast, Brown and colleagues [24] reported an increased whole-body fat oxidation rate during a 40-km cycling trial with 7-day AST supplementation (12 mg/day) in recreationally trained male cyclists. This increase in fat oxidation rate was actually recorded in the final stage of the cycling trial, where the VO₂max of the participants in their study was differed from that of the VO₂max of the participants in our study (VO_2peak: Brown et al. [24] = 56.5 ± 5.5 mL⋅kg^− 1⋅min^− 1; our study = 48.42 ± 8.21 mL⋅kg^− 1⋅min^− 1). We speculate that exercise intensity in our study may be the key factor influencing the effect of AST on energy substrate reliance during cycling challenge. The exercise intensity in our study was constant at 75% VO₂max during the cycling challenge, whereas Brown et al. adopted an exercise protocol with a graded intensity that started at 75.0 W and increased by 30.0 W every 1-min until volitional exhaustion [24]. Although AST enhanced endurance performance in our study, the effect of AST on fat oxidation might have been masked by the physiological adaptations associated with the intensity of exercise in young adults.

To further strengthen our findings, we tested the mood swings of participants after exhaustive exercise in both trials. We hypothesized that exhaustive cycling may induce certain mood disturbances in participants and that AST supplementation could attenuate such mood swings. The data on mood swings, including one positive (vigor) and five negative (tension, depression, anger, fatigue and confusion) domines, were obtained through the POMS. The scores of the variables were not altered by either a single bout of exhaustive exercise or AST supplementation. Among the five negative domains, the fatigue score was slightly lower in the AST trial but did not significantly differ from that in the placebo trial. The literature revealed that moderate exercise can improve the mood of participants, but such improvement does not occur after a single session of intense exercise. Instead, mood status can worsen following intense exercise compared to the mood state before exercise [51]. Regardless of exercise duration and recovery period, acute moderate-intensity running has been shown to have a favorable effect on the mood profile of young healthy adults [52]. Although natural AST has been demonstrated as an effective antidepressant in animal models [53], human studies are remains limited, particularly in the context of sports science. Therefore, available evidence are inconclusive to establish the beneficial effect of AST on the mood profile in adults following exercise challenge. Supplementation of natural AST at the dose of 12 mg/day for 8-week showed beneficial effects on overall mood, especially on decreasing of negative mood variables like depression and fatigue in healthy adults [30]. In another study, intake of AST for 8 weeks (12 mg/day) showed no effect on the POMS in adults; however, adults with strongly depressed symptoms might have experienced improved sleep quality after AST supplementation [54].

Limitations

Despite the strength of our findings on the ergogenic effects of AST, we have few limitations in this study. Changes in muscle damage, inflammatory or antioxidant markers were measured only during and immediately after exhaustive exercise, but not during post-exercise recovery. The absence of post-exercise recovery (24 to 72-h) assessments, particularly muscle damage markers (CK and LDH), limiting our understandings to state AST’s ability on attenuation of muscle damage. Long-term intervention with large sample size could provide statistical power to elucidate the anti-inflammatory and antioxidant efficiencies of AST during exercise challenge. Future studies with extended duration, large sample size and assessments during post-exercise recovery could further strengthen the ergogenic properties and clinical significance of AST.

Conclusion

Our findings demonstrated that short-term AST supplementation enhanced endurance performance among young college adults. Pre-supplementation of AST effectively decreased exhaustive cycling challenge-induced muscle damage and lipid peroxidation. The given dose and duration of AST (28 mg/day for 4-day, 112 mg in total) had no adverse effects on the mood states of the participants. These findings suggest that short-term AST supplementation can be considered a nutraceutical ergogenic aid to boost endurance performance.

Electronic supplementary material

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Abbreviations

AST

Astaxanthin

BMI

Body mass index

CK

Creatine kinase

hs-CRP

High-sensitivity C-reactive protein

LDH

Lactate dehydrogenase

MDA

Malondialdehyde

POMS

Profile of mood states

ROS

Reactive oxygen species

TTE

Time to exhaustion

TEAC

Trolox equivalent antioxidant capacity

TNF-α

Tumor necrosis factor-α

V̇O₂max

Maximum rate of O₂ uptake

Author contributions

Writing– original draft: Jung-Piao Tsao, Pei-Yu Wu; Writing– review & editing: Jung-Piao Tsao, Mallikarjuna Korivi, I-Shiung Cheng; Conceptualization: Pei-Yu Wu, Hsu-Tung Kuo; Methodology: Chih-Chieh Chen, Min-Yu Wang; Formal Analysis: Pei-Yu Wu, Hsu-Tung Kuo, Wei-Hsien Hong. All authors reviewed the manuscript.

Funding

This study was supported by grants from the National Science and Technology Council of Taiwan (grant no. NSTC 112-2410-H-039-001) and China Medical University (grant no. CMU 110-N-19).

Data availability

The data obtained from this study are included in the form of Tables and Figures. Further inquiries can be directed to the corresponding authors.

Declarations

Ethics approval and consent to participate

The study design and methodology were reviewed and approved by the Human Research Ethics Committee of China Medical University Hospital (CMUH111-REC3-081, Taichung, Taiwan), and registered at clinicaltrials.gov under registration number NCT06593535 (dated 05-09-2024). The study protocols were clearly explained to each participant, and written informed consent was obtained. The study was conducted in accordance with the ethical standards of the committee responsible for human experimentation, and with the Helsinki Declaration of 1975, as revised in 2013.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.