Effect of time-restricted feeding on cognitive dysfunction in Alzheimer’s disease and the role of ApoE polymorphism: protocol for a randomised controlled trial

Abstract

Introduction

With the global ageing population accelerating, the prevalence of Alzheimer’s disease (AD) continues to rise annually. However, the underlying mechanisms of AD remain unclear, and effective treatments are still lacking. Apolipoprotein E (ApoE) gene polymorphism and dietary habits are critical risk factors for AD. Time-restricted feeding (TRF), an intermittent fasting strategy that limits the daily window of food intake while maintaining nutritional balance, has garnered significant attention in recent years for its potential to improve cognitive dysfunction. This study aims to investigate the effect of TRF on cognitive improvement in AD patients within the context of genetic background and to explore the role of ApoE polymorphism in these mechanisms.

Methods and analysis

This single-centre, prospective, randomised, open-label, blinded-endpoint trial will recruit 160 patients with mild to moderate cognitive impairment due to AD from Peking University Shenzhen Hospital. Participants will be stratified based on ApoE genotype into ApoE4 non-carriers and ApoE4 carriers, then randomly assigned to either a TRF intervention group or a normal control group for a 24-month intervention period. The primary outcomes are changes in the Minimum Mental State Examination scale score, Montreal Cognitive Assessment scale score and Clinical Dementia Rating-Sum of Boxes scale score. Key secondary outcomes include the Activities of Daily Living scale score, blood AD biomarkers, lipid levels, ketone body levels and cerebral glutamate levels. Follow-up assessments will be conducted at baseline, 6, 12, 18 and 24 months.

Ethics and dissemination

Ethical approval for this study was obtained from the Research Ethics Committee of Peking University Shenzhen Hospital (Ethics No. 2024–151). Written informed consent will be obtained from all participants before the commencement of the trial. The findings will be disseminated through peer-reviewed publications.

Trial registration number

ChiCTR2400092653

STRENGTHS AND LIMITATIONS OF THIS STUDY.

• This study employs a stratified randomisation design based on apolipoprotein E ε4 carrier status, which will ensure balanced allocation across ε4 carriers and non-carriers, enhance the statistical power to detect genotype-specific treatment effects and allow for a more precise investigation of the mechanism of time-restricted feeding (TRF).

• We will undertake a comprehensive, multidomain assessment of efficacy, including detailed neuropsychological testing and biomarkers (eg, Aβ, τ), to provide a holistic evaluation of the intervention’s impact on Alzheimer’s disease (AD).

• This is a single-centre study that exclusively recruited participants from China.

• We limited the inclusion criteria to patients with mild to moderate AD, so the results may not be generalisable to individuals with severe AD.

• Due to the nature of the TRF intervention, the study was not blinded, but we controlled for bias by implementing blinding for outcome assessors.

Introduction

With the ongoing global ageing process, dementia has become a common disease among the elderly. A 2021 report published in The Lancet predicted that by 2050, the number of individuals with dementia will double worldwide.¹ Alzheimer’s disease (AD) stands out as the primary aetiological factor for dementia in the elderly, substantially contributing to the burden of disability and mortality within this age group.² Clinically, AD manifests through a progressive deterioration of memory, language, cognitive and behavioural abilities, significantly impairing both physical and mental health. The excessive aggregation of β-amyloid protein (Aβ) and the development of neurofibrillary tangles resulting from the hyperphosphorylation of Tau protein constitute the defining pathological hallmarks of AD. However, strategies to slow or prevent the clinical progression of AD remain elusive.^{3 4}

AD is influenced by multiple risk factors, with 60%–80% of the risk attributed to genetic factors.⁵ The apolipoprotein E (ApoE) allele stands out as the most potent genetic determinant associated with the development of AD.⁶ ApoE, a protein integral to lipid metabolism, is intricately implicated in lipid transportation and the repair processes following brain injury. ApoE has three isoforms—ApoE2, ApoE3 and ApoE4—encoded by three corresponding alleles.⁷ Compared with the protective effect of the ApoE2 allele, the ApoE4 allele has been identified as a substantial risk factor for AD. The presence of a single ApoE4 allele escalates the risk of developing AD by a factor of 3–4, whereas individuals carrying two ApoE4 alleles face an increased risk ranging from 12 to 15 times.8,10

Dietary interventions, such as adjusting dietary patterns, have been shown to mitigate the likelihood of cognitive decline and the emergence of dementia.¹¹ Recently, intermittent fasting (IF), a simple dietary approach to improving overall health, has gained increasing attention. Studies have demonstrated that fasting can reduce AD-related pathological hallmarks—including Aβ and phosphorylated τ—as well as attenuate microglial density and neuroinflammatory markers, thereby improving cognition in mouse models of AD.¹² Time-restricted feeding (TRF), a form of IF, involves limiting food intake to a time window each day. TRF is considered more moderate, easier to adhere to and more sustainable for the population at large.¹³

TRF confers significant metabolic benefits in humans, as evidenced by multiple clinical investigations. These effects have been documented in individuals with obesity,14,19 pre-diabetes,²⁰ type 2 diabetes,²¹ polycystic ovary syndrome,¹⁵ metabolic syndrome,²² non-alcoholic fatty liver disease,¹⁸ metabolic-associated fatty liver disease²³ and chronic kidney disease.²⁴ Specifically, TRF reduces body weight, enhances insulin sensitivity and attenuates systemic inflammation.^{14 15 18 20} Among various regimens, the 16:8 protocol (an 8-hour daily eating window) has demonstrated the most consistent benefits.1518 22 23 25,27 In specific populations—such as cancer patients undergoing radiotherapy—TRF may yield superior outcomes compared with conventional caloric restriction (CR).¹⁷

Notably, TRF has demonstrated a favourable safety profile and high adherence across diverse participant cohorts. Studies have shown that the regimen is well tolerated by healthy middle-aged and older adults,²⁷ older individuals at risk for overweight and mobility limitations,²⁸ as well as patients with moderate-to-severe chronic kidney disease,²⁴ with no serious adverse events (AEs) reported. Assessments using food diaries and similar tools have verified adherence rates exceeding 84%.^{21 28} Furthermore, the 16:8 protocol has been judged more feasible than alternate-day fasting (ADF) or more stringent eating-window restrictions.²⁹ These findings support the feasibility and sustainability of the 16:8 TRF paradigm in real-world settings, justifying its use in long-term clinical investigations.

However, existing clinical trials of TRF are generally limited by small sample sizes and short intervention periods, which restrict the generalisability of the findings and the evaluation of long-term efficacy. More importantly, direct clinical evidence investigating the effects of TRF on cognitive function in AD patients is currently lacking. Despite these limitations, studies on other IF regimens and preclinical experiments provide indirect support for its potential benefits. For example, 5:2 IF protocol has demonstrated positive effects on brain health in older adults.³⁰ Moreover, animal studies have directly shown that TRF attenuates AD-related pathology and improves cognitive performance in mouse models.³¹ During fasting, the brain shifts from glucose metabolism to ketone body metabolism, and ketone bodies produced during fasting positively regulate brain cell metabolism, mitochondrial function and other processes, activating lipid signals such as ApoE.³² Epidemiological evidence suggests that dietary patterns may interact with ApoE alleles, influencing AD development synergistically.³³

Given the absence of proven disease-modifying therapies for AD, TRF tailored to ApoE genotype represents a promising therapeutic avenue. We conducted a 16-week pilot study (n=6) to assess its feasibility in an AD population. The results demonstrated that the 16:8 TRF regimen was safe and well-tolerated, with high adherence (approximately 83%), and showed a potential for improving cognitive scores. These preliminary data formed the basis for the present full-scale, single-centre, prospective, parallel-group, open-label, randomised controlled trial (RCT) with blinded endpoints (PROBE design). A total of 160 patients with mild-to-moderate AD will be randomised in a 1:1 ratio to either the 16:8 TRF group or an ad libitum control group, under a superiority framework. Additionally, an exploratory arm comprising 80 healthy volunteers will provide normative baseline data. This trial aims to systematically evaluate the safety and efficacy of 16:8 TRF on cognitive function in AD patients. It will also examine whether ApoE polymorphisms modulate the intervention response and quantify changes in ketone metabolism and lipid profiles to elucidate potential mechanisms.

Methods and analysis

Study design

This is a single-centre, prospective, randomised, open-label trial with blinded endpoints, including baseline and follow-up assessments. Participants will be screened for mild-to-moderate cognitive impairment attributable to AD based on the diagnostic criteria established by the 2018 National Institute on Aging-Alzheimer’s Association (NIA-AA) guidelines, in conjunction with Minimum Mental State Examination (MMSE) scale score. A total of 160 patients will be enroled, with an additional 80 healthy volunteers recruited as a baseline control group.

Study population

The recruitment of potential participants will be meticulously orchestrated within the Department of Neurology at Peking University Shenzhen Hospital. The research team, in tandem with clinicians, will spearhead the recruitment and screening processes, ensuring that participants and their families are furnished with comprehensive written materials and detailed verbal elucidations regarding the study’s objectives, procedures and potential implications. The responsible study personnel will assist clinicians in assessing the severity of AD. Recruitment will be conducted in strict adherence to predefined inclusion and exclusion criteria, meticulously designed to ensure the homogeneity and suitability of the study population for the intended investigation. Prior to their involvement in the study, all participants and their family members will be required to thoroughly review the study information and provide their consent by signing an informed consent form (online supplemental material 1), underscoring the voluntary nature of participation and the right to withdraw at any juncture without incurring any adverse consequences.

Inclusion and exclusion criteria

Inclusion criteria

• Diagnosis of mild-to-moderate cognitive impairment attributable to AD, according to the 2018 NIA-AA criteria.

• Age ≥50 years, regardless of sex.

• Informed consent was obtained from the participant, with agreement to participate in the study and comply with all required visits, assessments and treatments as outlined in the protocol.

Exclusion criteria

• Severe AD and cognitive impairment due to surgery or other reasons.

• A history of inflammatory bowel disease, carbohydrate malabsorption, hormonal imbalance, known allergy to food additives or any other serious medical condition.

• History of gallbladder removal, gastrointestinal and cranial brain surgeries.

• Parasitic infections.

• Unable to cooperate with the study due to suicidal thoughts, attempts or aggressive behaviour.

• Use of medications known to affect gastrointestinal function, blood pressure and lipids, hormone supplements, allergy/asthma medications, proton pump inhibitors and over-the-counter medications.

• Use of probiotics or antibiotics or prebiotics (dietary fibre, oligosaccharides) within the 8 weeks prior to the trial, alcohol or drug abuse.

• Patients with a history of major trauma.

• Patients with moderate and severe malnutrition.

• Patients with type I diabetes, type II diabetes with fasting blood glucose greater than 10.11 mmol/L and latent autoimmune diabetes in adults.

• Use of drugs with known ketogenic tendencies (such as sodium-glucose cotransporter 2 (SGLT2) inhibitors, etc).

• Patients who have received an organ transplant or have previously received a non-autologous (allogeneic) bone marrow or stem cell transplant.

• Patients who have contraindications for cranial brain imaging such as post-steel plate placement, claustrophobia, etc, or inability to cooperate with perfect imaging examination because of agitation, etc.

Study visits

The visitation and intervention process of this study is depicted in figure 1. Participants are required to complete five follow-ups, spanning a total duration of 24 months. The overall duration of the study is planned for 5 years, from 31 October 2024 to 31 October 2029.

Figure 1. Study design and workflow of the study protocol. ApoE, apolipoprotein E; CEST MRI, Chemical Exchange Saturation Transfer MRI; NC, normal control; TRF, time-restricted feeding.

Baseline visit

The clinical investigators will screen potential participants for eligibility and obtain written informed consent.

Baseline data will be collected, including demographic information, medical history, physical examination findings and medication usage.

Further data on fasting blood glucose, blood pressure, heart rate, urine routine, liver and kidney function will be collected as safety indicators.

ApoE genotyping will be performed to determine whether participants are ApoE4 non-carriers (ApoE4^-/-) or carriers (ApoE4^+/-/ApoE4^+/+).

Cognitive function will be assessed using the MMSE, Montreal Cognitive AssessmentMontreal Cognitive Assessment (MoCA) and Clinical Dementia Rating-Sum of Boxes (CDR-SB) scales.

Activities of daily living (ADL) will be assessed using the ADL scale.

Fasting blood samples will be collected to examine a range of biomarkers, including peripheral blood AD markers (Aβ40, Aβ42, p-Tau181, p-Tau217, NFL (neurofilament light chain)), lipid levels (total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C)) and serum ketone bodies.

Additional blood, urine, faecal and skin patch samples will be collected for future metabolomics studies.

Faecal samples will also be collected for future gut microbiota research.

All participants will undergo chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) to quantify cerebral glutamate levels. The detailed imaging protocol is as follows:

Scanning will be performed using a United Imaging Jupiter 5T MRI system equipped with a dedicated head-neck coil. The acquisition protocol includes:

Conventional T1- and T2-weighted structural sequences (acquisition time: ~10 min) and magnetic resonance spectroscopy (~20 min); (2) whole-brain three-dimensional CEST imaging (~10 min).

Study intervention

AD participants will be randomised into four parallel groups in a 1:1 ratio based on ApoE genotype: (1) ApoE4 non-carrier+normal diet (ApoE4^-/- + NC (Normal diet)). (2) ApoE4 non-carrier+TRF (ApoE4^-/- + TRF). (3) ApoE4 carrier+normal diet (ApoE4^+/-/ApoE4^+/+ + NC). (4) ApoE4 carrier+TRF (ApoE4^+/-/ApoE4^+/+ + TRF). Healthy volunteers will serve as a baseline control group. The first and third groups will follow a normal diet, while the second and fourth groups will receive the TRF intervention.

To help participants adapt to the intervention and reduce potential lifestyle differences before randomisation, a 1-week induction phase will be implemented. During this phase, all participants will receive dietary and lifestyle guidance through expert lectures, printed educational materials and WeChat posts (based on the 2016 Chinese Dietary Guidelines and the 2017–2025 National Healthy Lifestyle Action Plan). After the induction period, participants will be randomised into either the NC or TRF group.

In the TRF group, participants are required to consume food within a designated time window each day, without restrictions on the food content or nutrient proportions. Available time windows for food intake are 08:00–16:00, 09:00–17:00 or 10:00–18:00. Participants may divide their food intake into any number of main meals or snacks, with the remaining time being fasting hours. During the fasting period, no caloric food or beverages are allowed (except black coffee or unsweetened tea). Participants must maintain the TRF pattern for at least 5 days per week, with the total intervention duration lasting 24 months. Participants will be encouraged to drink sufficient water throughout the intervention. The TRF intervention will be discontinued and return to a normal diet if participants experience 50% weight loss or sustained weight loss for 12 weeks (whichever occurs first). Participants may continue all usual medications. No concomitant treatments are prohibited; any new prescription or supplement will be recorded in the CRF for post hoc sensitivity analysis.

To quantify and report adherence, we developed the following monitoring strategies: (1) structured digital logging and real-time supervision: To achieve high-frequency monitoring of daily behaviour, we have established individualised WeChat groups for each participant (comprising the clinical researcher, nutritionist, participant and one family member). Participants are required to report their first and last meal times daily via text message in the group and report their fasting body weight weekly. Furthermore, we have created a password-protected online shared document where participants are instructed to log their detailed timestamps of daily eating events and sleep periods. (2) Objective indicator verification: our protocol required measurement of the serum ketone bodies and lipid levels during every scheduled clinical follow-up visit.

To enhance adherence among participants at risk of cognitive impairment, we will simplify the dietary protocol for each participant. We will work with participants and their caregivers to define an individualised 8-hour eating window that aligns with their existing lifestyle and routines (08:00–16:00, 09:00–17:00 or 10:00–18:00). A family member or primary caregiver is integrated into the compliance strategy. They are included in the WeChat group, receive education on the study’s importance, and are empowered to provide daily reminders, prepare meals within the window and offer crucial encouragement. Beyond the daily WeChat check-ins, the research team (including the nutritionist) will provide weekly positive feedback based on the shared logs. We will closely monitor for any adverse effects (eg, excessive fatigue, dizziness, unwanted weight loss) during follow-up calls and visits. If such issues arise, the protocol can be temporarily relaxed or personalised (eg, widening the eating window to 10 hours) to retain the participant in the study while still capturing meaningful adherence data. Regular measurement of blood ketone levels provides an objective biological marker that corroborates the self-reported fasting data, allowing us to identify and address non-compliance objectively.

Subsequent clinical reviews

To ensure participant safety during the study, frequent follow-up visits will be conducted. Follow-up assessments will take place at baseline (0 months), 6 months, 12 months, 18 months and 24 months.

Study endpoints

Primary outcome

The primary outcomes include the MMSE scale scores, the MoCA scale scores and the CDR-SB scale scores. These outcomes will be assessed at baseline (0 months), 6 months, 12 months, 18 months and 24 months.

Secondary outcomes

The secondary outcomes of the study include the ADL scale scores, peripheral blood AD biomarkers (Aβ40, Aβ42, p-Tau181, p-Tau217, NFL), changes in serum lipid levels (TC, TG, LDL-C, HDL-C) before and after the intervention, changes in serum ketone bodies before and after the intervention, and additional markers including serum metabolomics, gut microbiota and cerebral glutamate levels.

Safety

AEs in this study are defined as mild clinical symptoms such as fatigue, weakness and dizziness. Researchers will closely monitor any AEs (including adverse reactions or unforeseen symptoms, signs and laboratory findings) throughout the study. Clinical nutritionists will initially assess the severity of AEs and determine whether to continue the intervention. Any AE reported by participants during the intervention period will be documented and reported in real-time to the project team. All AEs will be reviewed within 3 days to confirm whether they have improved. If no improvement is observed, the clinical expert team will decide whether to discontinue the intervention for the participant. Safety will be monitored throughout the study via AE logs, which will document the timing, severity, interventions, treatment relatedness and time to recovery for each event.

Sample size

Sample size estimation was performed using the formula n = [Uα + Uβ]/(P experimental – P control), where n represents the number of participants per group. The estimated sample size is 33 per group, and considering potential dropout, a 10%–20% increase was applied, resulting in 40 participants per group.

Randomisation and blinding

Participants carrying or not carrying the ApoE4 genotype were randomly assigned to two groups using a randomisation method with a block size of 4. The randomisation sequence was generated by an independent statistician using a computerised random number generator. The allocation ratio between the TRF intervention group and the control group was 1:1. To ensure allocation concealment, the randomisation list was sealed in sequentially numbered, opaque envelopes, which were opened only after participants had been screened and deemed eligible for enrolment. Randomisation was performed by an independent staff member who was not involved in the clinical study. Given that TRF requires participant cooperation, this trial was conducted as an open-label study. To control for bias and minimise variability in outcome assessment, blinding was specifically applied to the outcome assessors. No emergency unblinding mechanism is required since knowledge of the allocated eating window is not clinically critical.

Consent process

The research team will ensure that both participants and their legal representatives fully understand the study's purpose, procedures, associated risks, and potential benefits. It will be explicitly communicated to participants that they retain the prerogative to disengage from the study at their discretion, free from any repercussions. Prior to their engagement in the study, all participants are mandated to furnish written consent. In instances where a participant is deemed incapable of granting consent due to legal incapacitation, the procurement of written consent from a designated legal custodian is imperative.

Data management and confidentiality

Each participant will be obligated to fill out an initial data sheet and a clinical case report form (CRF), both of which will serve as the foundation for constructing an electronic data repository. Researchers will complete the CRF during patient treatment, ensuring data are recorded in a timely, complete, accurate and truthful manner. Any corrections to the CRF will be made by striking through the original data and writing the corrected information next to it, with the researcher’s signature and date. Prior to the clinical trial, all researchers will undergo training to ensure they fully understand the study protocol and the specific measures involved. Symptom and sign quantification will undergo consistency checks. Laboratory data will be documented within the CRF, with the original reports appended to the patient’s medical files. Values that deviate from the clinically permissible range will be authenticated, and attending physicians will offer requisite elucidations. Data entry into the electronic platform will be double-checked. All study records, including signed informed consent forms and CRFs, will be retained for 10 years. Electronic records will be safeguarded through encryption and password protection, while physical data storage will be secured in a restricted area accessible solely to authorised individuals. Data amassed throughout the study will not be included in the clinical records and will be anonymised to prevent disclosure of personal information. Researchers engaged in the trial will gain access to the ultimate dataset on the trial’s completion, which will be available at the conclusion of the trial on reasonable request.

Handling of missing data

Strategies to minimise attrition include flexible visit scheduling, regular reminders via the WeChat platform, active caregiver engagement and the collection of at least two alternative contact methods. Missing data will be handled according to the intention-to-treat principle. The primary analysis will employ Multiple Imputation by Chained Equations to create 20 imputed datasets under the Missing at Random assumption. The imputation model will incorporate the outcome trajectory, treatment group assignment and prespecified covariates. To assess robustness, sensitivity analyses will be conducted, including complete-case analysis and extreme-scenario imputation.

Statistical analysis

Data will be conducted using SPSS software, specifically V.22.0. All statistical tests will be executed as two-tailed, with a significance threshold set at p<0.05 to determine statistical significance. The estimation of CIs for various parameters will be based on 95% CIs. Continuous data will be depicted either as means±SD or as medians accompanied by IQR, and categorical data will be expressed as percentages. Comparisons between baseline and postintervention groups will use either unpaired t-tests or Mann-Whitney U tests, depending on whether the continuous data follow a normal distribution. Within-group comparisons of pre- and postintervention data will be performed using paired t-tests or Wilcoxon tests, depending on normality. Categorical data will be analysed using χ² tests. Mixed linear regression will be used to assess intervention effects, time effects and the interaction between intervention and time.

To control for potential confounding, we will prospectively collect data on key prognostic factors, including age, sex, years of education, baseline MMSE score, ApoE ε4 status and cardiovascular risk factors (eg, systolic blood pressure, HbA1c). Balance of these covariates postrandomisation will be assessed using standardised mean differences (<0.10 will indicate good balance). The primary analysis will use an analysis of covariance model, which will mandatorily adjust for the baseline value of the outcome measure, age and years of education. Additionally, we will report a sensitivity analysis from a fully adjusted model that includes all prespecified covariates to demonstrate the robustness of our findings.

Monitoring

All study personnel, including clinicians, outcome evaluators, data gatherers, data supervisors, data input operators and statistical analysts, will undergo targeted instruction regarding standardised operational protocols and data administration. Participant data will be recorded on CRFs and independently entered into an Excel database by two data entry personnel. The data manager will cross-check the two datasets for accuracy. Given the relatively small sample size of this trial, an independent Data Monitoring Committee was not established. No interim analyses or stopping rules are planned because the intervention is low-risk. No independent audit is planned because this is a single-centre, low-risk dietary intervention.

Patient and public involvement

None.

Discussion

AD is the most prevalent neurodegenerative disorder globally, with early symptoms including a decline in learning and memory, ultimately leading to severe memory and behavioural impairments.⁴ Although a substantial amount of research has been conducted, the precise aetiology of AD is still not fully elucidated. Presently, clinical therapeutic approaches are only capable of mitigating the advancement of symptoms to a limited extent.³⁴ As such, there is an urgent need for additional non-pharmacological interventions to complement existing therapeutic approaches for AD.

In recent years, IF has emerged as a widely studied non-pharmacological intervention, showing promising effects on conditions such as diabetes, obesity and age-related diseases.³⁵ Studies on ADF and similar prolonged fasting regimens have demonstrated improvements in spatial memory and a reduction in brain Aβ accumulation in AD mouse models,³⁶ however, these fasting methods are often extreme, have a narrow applicable population, are difficult to sustain, and carry risks such as dehydration and malnutrition.^{37 38} As a result, more moderate and sustainable fasting approaches, such as TRF, have gained increased attention in both clinical and basic research.

A 2019 review published in The New England Journal of Medicine highlighted the positive impact of TRF and other restricted feeding strategies on lifespan extension and reduced risk of age-related diseases.³⁵ Basic studies have shown that TRF improves cognitive performance and spatial working memory in mouse models of vascular cognitive impairment,³⁹ and significantly enhances memory and cognitive function while reducing Aβ deposition in AD rat models.⁴⁰ Several clinical studies have been conducted in cognitively healthy individuals. One study in Italy found a significant positive correlation between TRF and improved cognitive status in older adults.⁴¹ However, a RCT involving 16-hour fasting for 4 weeks did not find significant cognitive improvements in healthy elderly individuals.²⁸ Another study revealed no notable changes in interleukin-6 (IL-6), oxidised LDL or ketone body levels after 6 weeks of TRF intervention (16-hour fasting).²⁷

For individuals with mild cognitive impairment (MCI), two studies have reported potential cognitive benefits from CR and IF. In a prospective controlled trial involving elderly, obese individuals with MCI, a 12-month CR intervention reduced BMI and was correlated with enhancements in verbal recall, verbal fluency, executive capabilities and overall cognitive scores, with the strongest effects observed in ApoE4 carriers.⁴² Another study involving 99 elderly individuals with MCI who underwent long-term IF (fasting on Mondays and Thursdays) showed higher cognitive scores after 36 months of follow-up.⁴³ Further metabolomic analyses suggested that IF could modulate cognitive function via a multitude of metabolic pathways, encompassing the synthesis and degradation of ketone bodies, butyrate metabolism, pyruvate metabolism, as well as the glycolysis and gluconeogenesis pathways. Despite these findings, randomised controlled clinical studies on the relationship between TRF and AD-related cognitive dysfunction remain limited, and current studies often have small sample sizes. The effects of TRF on improving cognitive function in AD along with the associated underlying mechanisms remain inadequately elucidated.

AD risk factors include both genetic and dietary factors.⁴⁴ Among the genetic factors, the ApoE gene stands out as the most prominent recognised risk factor for AD.⁴⁵ The amino acid polymorphisms between different ApoE alleles affect their affinity for ApoE receptors and lipid clearance efficiency, leading to variations in disease incidence.⁴⁶ Under typical physiological states, ApoE is predominantly released by astrocytes within the brain and undergoes lipidation via ATP-binding cassette A1 transporter on cell membranes.⁴⁷ However, different ApoE isoforms have been shown in animal models to influence Aβ pathology and lipidation differently. Compared with ApoE2 and ApoE3, ApoE4 exhibits weaker stability in complexing with Aβ.⁴⁸ During fasting periods greater than 12 hours, blood glucose levels and liver glycogen stores decline, and the body shifts from relying on glucose derived from glycogen breakdown to using fatty acids released from fat stores.⁴⁹ These fatty acids are metabolised into ketone bodies by the liver, which are then distributed to other cells, especially in the brain.⁵⁰

However, there is limited research that distinguishes participants by ApoE genotype to explore the effects of fasting on improving AD. Additionally, a deficiency of rigorous analysis persists in this domain. The objective of this study is to examine the effects of TRF on cognitive function in individuals with AD through a randomised controlled clinical trial. Furthermore, based on ApoE genotyping, AD patients will be classified into ApoE4 carriers or non-carriers to assess differences in cognitive improvements between the two groups following TRF intervention. This study will also combine multidisciplinary techniques to explore how TRF modulates ketone body and lipid metabolism in AD patients and the mediating role of ApoE isoforms in these processes. However, the primary outcome measure of this study is cognitive dysfunction scores, and the main focus will be on examining the impact of TRF on AD-induced cognitive dysfunction during follow-up. Additionally, the study will include only patients with mild to moderate AD, meaning the results may not be applicable to individuals with severe AD. Because of the nature of the TRF intervention, blinding for the participants is not feasible, but to control for bias, outcome assessors will be blinded. Although blinding of endpoints helps minimise bias during outcome evaluation, limitations remain in controlling for information bias resulting from subjective factors of both the researchers and participants.

Ethics and dissemination

This clinical trial will adhere to the ethical, scientific and legal principles outlined in the Declaration of Helsinki and the Interim Measures for the Administration of Human Genetic Resources of China. The study received ethical approval from the Research Ethics Committee of Peking University Shenzhen Hospital (2024–151, 30 October 2024). This trial was registered with the Chinese Clinical Trial Registry (ChiCTR) on 21 November 2024 (ChiCTR2400092653).

Before the study commences, the study protocol, any amendments and the informed consent form must receive written approval or a favourable opinion from the Ethics Committee. Researchers are responsible for the medical care of participants, making any clinical decisions related to the study and ensuring that appropriate management or treatment is provided in the event of any AEs during the study. Ancillary care and compensation for trial-related injury are described in the participant consent form (online supplemental material 1). To protect participants’ privacy, all CRFs, study reports and communications related to the research will use participant identification codes rather than personal names. It is anticipated that data collection will be completed by the end of October 2028. The study results will be submitted for publication within 6 months of database lock, and there are no restrictions on publication. De-identified participant-level data, the full protocol and statistical code will be made available on reasonable request to the corresponding author.