Fasting, Exercise, and Diet to Activate Autophagy in Depression (AutoFast)

Targeting Autophagy in Depression: Fasting, Exercise, Diet

Depression is a common mental health condition that affects millions of people worldwide and is a leading cause of disability. Although current treatments can be effective, many patients do not fully recover or experience long-term improvement. This study aims to better understand how lifestyle factors such as physical activity and diet-related processes may influence biological mechanisms that could be linked to depression.

The study focuses on a natural cellular process called autophagy, which helps cells remove damaged components and maintain healthy function. Autophagy is influenced by energy availability in the body and may be affected by behaviors such as physical exercise and caloric restriction. Early evidence suggests that changes in autophagy may also be linked to mood regulation and depression, but this relationship is not yet well understood in humans.

In this exploratory study, we will investigate how physical activity influences autophagy and related metabolic and molecular processes in healthy adults. We will also examine whether these effects differ between individuals with different body weight and fitness levels, and between women and men.

A total of approximately 120 healthy adults aged 18 to 40 years will participate. Participants will be divided into four groups based on sex and body weight (normal weight or overweight). Each participant will attend study visits at the University Hospital Zurich and perform a standardized cycling exercise test under medical supervision.

During the exercise test, participants will perform a graded cycling protocol that gradually increases in intensity until exhaustion. We will collect small blood samples from a vein and from a fingertip at several time points before, during, and after exercise. Saliva samples will also be collected to measure stress-related hormones. Additional measurements include heart rate, breathing parameters, oxygen consumption, and physical performance.

Blood and saliva samples will be analyzed using advanced laboratory techniques to study changes in metabolism, immune signaling, hormones, gene activity, and markers related to autophagy. These analyses will help identify biological pathways that are activated by exercise and may be relevant to brain health and depression.

Participants will undergo medical screening before inclusion to ensure safety. Individuals with certain medical conditions or factors that could interfere with the study results will not be included. Participation is voluntary, and participants may withdraw at any time without consequences.

The study involves minimal risks associated with blood sampling and intense physical exercise, which will be performed under close medical supervision. The expected benefit is improved scientific understanding of how lifestyle-related biological processes may be linked to mental health, which could support the development of new preventive or therapeutic strategies for depression in the future.

Study Overview

• Status: Not yet recruiting
• Conditions: Depression - Major Depressive Disorder; Overweight (BMI > 25)
• Intervention / Treatment: Procedure: Performance test

Detailed Description

Background and Rationale Depressive disorders are among the leading causes of disability worldwide and represent a major public health burden. Despite the availability of pharmacological and psychotherapeutic treatments, a substantial proportion of patients do not achieve full remission or experience relapse. Current antidepressant strategies primarily target monoaminergic systems and are often insufficient in addressing the biological heterogeneity of depression.

Emerging evidence suggests that metabolic regulation and cellular stress response pathways may play an important role in the pathophysiology of depression. In particular, associations between metabolic disorders (such as obesity and insulin resistance) and depressive symptoms indicate shared biological mechanisms. This has led to increasing interest in lifestyle-based interventions, including physical activity, dietary modification, and caloric restriction, as potential modulators of both metabolic and neuropsychiatric outcomes.

A central candidate mechanism linking metabolism and brain function is autophagy, a conserved cellular process responsible for the degradation and recycling of damaged proteins and organelles. Autophagy is tightly regulated by nutrient availability and energy status, primarily via the AMPK-mTOR signaling axis. It is activated under energy deprivation and suppressed under nutrient excess. Proper autophagic flux is essential for neuronal homeostasis, immune regulation, and cellular stress adaptation.

Preclinical and emerging clinical evidence suggests that impaired autophagy may be involved in psychiatric disorders, including depression. Furthermore, interventions such as physical exercise, caloric restriction, and certain pharmacological agents have been shown to modulate autophagy-related pathways. However, the direct measurement of autophagic flux in humans under physiological conditions remains methodologically challenging, and its relationship to exercise-induced metabolic and neurobiological changes is not fully understood.

This study aims to address this gap by investigating autophagy-related biological responses to acute physical exercise in humans using a multi-omics approach.

Objectives Primary Objective To investigate whether acute physical exercise induces measurable changes in autophagy-related pathways and associated metabolic, proteomic, transcriptomic, and hormonal markers in humans.

Secondary Objectives To characterize exercise-induced changes in systemic metabolism, inflammatory markers, and stress hormones.

To explore associations between fitness level, body mass index (BMI), and molecular responses to exercise.

To identify potential biomarkers of autophagy activation in peripheral blood and saliva.

To generate mechanistic hypotheses linking metabolic regulation, autophagy, and pathways relevant to mood disorders.

Study Design This is a single-center, exploratory human research study conducted at the University Hospital Zurich in collaboration with exercise physiology facilities.

The study uses a cross-sectional experimental design involving standardized acute exercise stimulation (cardiopulmonary exercise testing, CPET) combined with repeated biological sampling and multi-omics profiling.

Participants will be stratified into four groups based on sex and BMI:

Normal-weight women Normal-weight men Overweight women Overweight men All participants will perform a standardized incremental cycling exercise test under controlled laboratory conditions.

Study Population Approximately 120 healthy adults aged 18-40 years will be included. Participants will be selected based on predefined inclusion and exclusion criteria to ensure medical safety and reduce confounding variables such as chronic disease, medication use, psychiatric disorders, and hormonal influences (e.g., hormonal contraception or pregnancy in women).

Women will be tested during early follicular phase (cycle days 1-5) to minimize hormonal variability.

Study Procedures

Each participant will undergo:

1. Screening and Baseline Assessment Informed consent Medical history and physical screening Assessment of inclusion/exclusion criteria Questionnaires assessing mood, anxiety, and physical activity Serological screening for HIV and hepatitis B/C
2. Physiological Measurements (Pre-exercise) Body composition analysis (DXA) Lung function testing Baseline blood sampling Saliva sampling for cortisol
3. Exercise Intervention (CPET) Participants will perform a standardized graded cycling exercise test on an electromagnetically braked ergometer.

The protocol includes:

15-minute warm-up phase at submaximal intensity Incremental ramp protocol until voluntary exhaustion

Continuous monitoring of:

Oxygen uptake (VO₂) Carbon dioxide production (VCO₂) Heart rate and ECG Blood pressure Respiratory exchange ratio

Key physiological thresholds will be determined:

Aerobic threshold Anaerobic threshold Respiratory compensation point 4. Biological Sampling

Repeated biological sampling will be performed at defined time points:

Rest (baseline) End of warm-up (aerobic phase) Peak exercise (maximal exertion) 10 minutes recovery 30 minutes recovery

Samples include:

Venous blood (PBMC isolation and plasma) Capillary blood microsamples (fingertip sampling devices) Saliva (cortisol analysis) Urine (pregnancy test in women) Total blood volume per participant will be approximately 320 mL across all time points.

Laboratory Analyses

Collected samples will be used for multi-layered molecular profiling:

1. Autophagy-Related Analyses LC3B-II-based flux assays in PBMCs Ex vivo stimulation assays with lysosomal inhibition (chloroquine-based approach) Quantification of autophagy-related proteins (e.g., ATG family, ULK1 pathway components) Gene expression profiling of autophagy signaling pathways
2. Metabolomics and Lipidomics Targeted and untargeted metabolomic profiling Energy substrates and oxidative stress markers Polyamine metabolism (e.g., spermidine-related pathways) Steroid hormone profiling via mass spectrometry
3. Proteomics and Transcriptomics Plasma and PBMC proteomic profiling (untargeted and targeted) Phosphoproteomic analysis Single-cell or bulk RNA sequencing of immune cells
4. Inflammatory and Immune Markers Cytokine quantification (e.g., IL-1β, IL-6, IL-10, TNF-α) Markers of immune activation and systemic inflammation
5. Hormonal and Stress Response Measures Cortisol (saliva and plasma) Sex steroid hormones (e.g., estradiol, testosterone, progesterone) Hypothalamic-pituitary-adrenal (HPA) axis-related markers
6. Genomic and Epigenetic Analyses DNA damage mapping (e.g., oxidative lesions, strand breaks) DNA methylation profiling (EPIC array) Gene regulation changes in response to exercise-induced stress

• Study Type: Interventional
• Enrollment (Estimated): 120
• Phase: Not Applicable

Contacts and Locations

Study Locations

• Switzerland
  • Canton of Zurich
    • Zurich, Canton of Zurich, Switzerland, 8091
      • University Hospital Zurich
      • Contact: Jens Stepan, MD, PhD; Phone Number: +41 43 253 17 59; Email: jens.stepan@usz.ch
      • Principal Investigator: Jens Stepan, MD, PhD

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult
• Accepts Healthy Volunteers: Yes

Description

Inclusion Criteria:

• age: 18-40 years
• BMI: between 18.5 and 24.9 kg/m2 (SG1/2) or BMI between 25.0 and 39.9 kg/m2 (SG3/4)
• ability to understand the study procedure and give consent
• Written informed consent
• SG1 women: any fitness level
• SG2 men: VO2max < 45 ml/kg/KG29,30
• Available to conduct CPET on menstrual cycle days 1-5 (SG1/3)
• No infection with HIV or Hepatitis B/C

Exclusion Criteria:

• No infectious illness for at least two weeks prior to the test
• No vitamin supplementation during the week prior to the performance test
• SG3 and SG4: More than 1 hour moderate exercise per week
• No use of hormonal contraceptives in the last 6 months before the onset of the study (SG1/3)
• a clinically diagnosed menstrual disorder (e.g., polycystic ovarian syndrome or amenorrhea) (SG1/3)
• having given birth within the 12 months before inclusion in the study (SG1/3)
• pregnancy or breastfeeding (SG1/3)
• premenstrual dysphoric disorder (PMDD) (SG1/3)
• history of epileptic seizure
• history of depression
• history of manic or psychotic episode
• existing/current eating disorders (bulimia nervosa, anorexia nervosa) within the past 5 years
• inability to communicate adequately in speech
• inability to follow instructions
• regular use of medication other than thyroxine
• alcohol consumption as equivalent doses of more than 12 g of pure alcohol per day on average for women and 24 g of pure alcohol per day for men
• vegan diet
• daily nicotine consumption
• currently or history of (regular) consumption of illegal drugs within the last year
• known diseases of the cardiovascular system
• arterial hypertension above 160/90 mmHg at rest
• known pulmonary diseases
• arthritis and rheumatic diseases and conditions
• hematologic diseases
• bronchial asthma
• surgery less than 4-6 months ago
• orthopedic or other diseases (e.g. neurological) that preclude maximum load on the bicycle ergometer
• anemia (<12.0 g/dl for women and <14.0 g/dl for men)

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Non-Randomized
• Interventional Model: Parallel Assignment
• Masking: None (Open Label)

Number of Arms

4

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Healthy, normal weight women, regular menstrual cycle, no hormones, all fitness levels	Procedure: Performance test; Standardized CPET on a bicycle ergometer. Venous and capillary blood sampling at baseline (rest), end of aerobic warm-up phase (15 min), peak exercise (ramp-protocoll, 8-12 min), 10 minutes post-exercise, and 30 minutes post-exercise.
Experimental: Healthy normal weight men, recreational fitness level	Procedure: Performance test; Standardized CPET on a bicycle ergometer. Venous and capillary blood sampling at baseline (rest), end of aerobic warm-up phase (15 min), peak exercise (ramp-protocoll, 8-12 min), 10 minutes post-exercise, and 30 minutes post-exercise.
Experimental: Healthy, overweight women, regular menstrual cycle, no hormones, recreational fitness levels	Procedure: Performance test; Standardized CPET on a bicycle ergometer. Venous and capillary blood sampling at baseline (rest), end of aerobic warm-up phase (15 min), peak exercise (ramp-protocoll, 8-12 min), 10 minutes post-exercise, and 30 minutes post-exercise.
Experimental: Healthy, overweight men, regular menstrual cycle, no hormones, recreational fitness levels	Procedure: Performance test; Standardized CPET on a bicycle ergometer. Venous and capillary blood sampling at baseline (rest), end of aerobic warm-up phase (15 min), peak exercise (ramp-protocoll, 8-12 min), 10 minutes post-exercise, and 30 minutes post-exercise.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Autophagic Flux in Peripheral Blood Mononuclear Cells (PBMCs)	Assessment of autophagic flux in PBMCs using ex vivo lysosomal inhibition and quantification of autophagy-related proteins (e.g., LC3B-II) to evaluate exercise-induced activation of autophagy pathways.	Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise (single study day).

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
PBMC Transcriptome	Genome-wide transcriptomic profiling of PBMCs using RNA sequencing to identify exercise-induced changes in gene expression and cellular signaling pathways.	Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.
PBMC Proteome	Untargeted proteomic analysis of PBMCs to characterize exercise-induced changes in protein abundance and biological pathways.	Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.
PBMC Phosphoproteome	Untargeted phosphoproteomic profiling of PBMCs to assess exercise-induced changes in intracellular signaling and protein phosphorylation.	Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.
Plasma Proteome	Untargeted plasma proteomics to identify circulating proteins and pathways altered by acute physical exercise.	Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.
Targeted Plasma Metabolomics	Quantification of metabolites involved in energy metabolism, autophagy regulation, oxidative stress, and exercise adaptation using targeted metabolomic approaches.	Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.
Untargeted Metabolomic Profile	Comprehensive metabolomic profiling to identify exercise-induced changes in metabolic pathways associated with autophagy, energy homeostasis, and stress responses.	Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.
Polyamine Concentrations	Quantification of circulating polyamines and related metabolites, including spermidine-associated pathways implicated in autophagy regulation.	Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.
Steroid Hormone Profile	Measurement of circulating steroid hormones, including cortisol, cortisone, DHEA, DHEAS, progesterone, estradiol, testosterone, aldosterone, and related metabolites using mass spectrometry.	Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.
Inflammatory Marker Profile	Quantification of circulating inflammatory cytokines and immune mediators, including IL-1α, IL-4, IL-6, IL-10, IL-13, and TNF-α.	Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.
Genome-wide DNA Oxidation Profile	Genome-wide mapping of oxidative DNA lesions in PBMCs to assess exercise-induced oxidative stress and genomic responses.	Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.
Genome-wide DNA Strand Break Profile	Genome-wide assessment of DNA strand breaks in PBMCs to characterize genomic stress responses induced by acute exercise.	Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.
DNA Methylation Profile	Genome-wide DNA methylation analysis to investigate exercise-induced epigenetic regulation and associations with transcriptional responses.	Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.
Extracellular Vesicle Profile	Characterization of circulating extracellular vesicles and their molecular cargo as potential mediators of exercise-induced intercellular communication.	Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.
Cardiorespiratory Fitness (VO₂max)	Maximum oxygen uptake measured during cardiopulmonary exercise testing as an indicator of aerobic fitness and exercise capacity.	Measured during the exercise test on the study day.
Blood Lactate Concentration	Capillary blood lactate concentrations measured during exercise to assess metabolic responses and anaerobic metabolism.	Baseline (rest), end of aerobic warm-up phase, and peak exercise.
Body Composition	Assessment of body fat mass, lean mass, and bone mineral density using dual-energy X-ray absorptiometry (DXA).	Baseline assessment prior to exercise testing.
Depressive Symptoms (Beck Depression Inventory-II, BDI-II)	Assessment of depressive symptom severity using the Beck Depression Inventory-II (BDI-II), a validated self-report questionnaire.	Baseline (Visit 1, prior to exercise testin
Anxiety Symptoms (State-Trait Anxiety Inventory, STAI)	Assessment of state and trait anxiety using the State-Trait Anxiety Inventory (STAI).	Baseline (Visit 1, prior to exercise testing).
Physical Activity Level (International Physical Activity Questionnaire, IPAQ)	Assessment of habitual physical activity and exercise behavior using the International Physical Activity Questionnaire (IPAQ).	Baseline (Visit 1, prior to exercise testing).
Positive and Negative Affect (PANAS)	Assessment of positive and negative affective states using the Positive and Negative Affect Schedule (PANAS).	Immediately before exercise testing and 30 minutes after completion of exercise testing.
Mood States (Profile of Mood States, POMS)	Assessment of transient mood states, including tension, depression, anger, vigor, fatigue, and confusion, using the Profile of Mood States (POMS).	Immediately before exercise testing and 30 minutes after completion of exercise testing.
Subjective Well-Being (Befindlichkeitsskalen, BF)	Assessment of current subjective well-being and psychological state using the Befindlichkeitsskalen (BF).	Immediately before exercise testing and 30 minutes after completion of exercise testing.
Current Psychological State (Eigenzustandsskala, EZ)	Assessment of participants' current psychological state using the Eigenzustandsskala (EZ).	Immediately before exercise testing and 30 minutes after completion of exercise testing.
Premenstrual Symptoms (PMS Questionnaire)	Assessment of menstrual cycle-related symptoms in female participants using a standardized Premenstrual Syndrome (PMS) questionnaire.	Baseline (Visit 1, female participants only).
Capillary Blood Microsampling	Evaluation of two certified capillary blood microsampling systems (Mitra® device and Whatman® 903 Protein Saver cards) for the collection of capillary blood samples. Comparisons will assess sample quality, analytical performance, feasibility, and concordance of omics-based measurements obtained from microsamples across repeated exercise-associated sampling time points.	Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.

Collaborators and Investigators

• Sponsor: University of Zurich
• Collaborators: University Hospital, Bonn; ETH Zurich

Publications and helpful links

General Publications

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Study record dates

Study Major Dates

• Study Start (Estimated): September 11, 2026
• Primary Completion (Estimated): September 30, 2029
• Study Completion (Estimated): September 30, 2029

Study Registration Dates

• First Submitted: June 17, 2026
• First Submitted That Met QC Criteria: June 17, 2026
• First Posted (Actual): June 24, 2026

Study Record Updates

• Last Update Posted (Actual): June 24, 2026
• Last Update Submitted That Met QC Criteria: June 17, 2026
• Last Verified: June 1, 2026

More Information

Terms related to this study

• Keywords: depression; metabolomics; autophagy; proteomics; steroid hormones; multi-omics; performance testing; autophagy flux
• Additional Relevant MeSH Terms: Nutrition Disorders; Overnutrition; Body Weight; Behavioral Symptoms; Pathological Conditions, Signs and Symptoms; Behavior; Nutritional and Metabolic Diseases; Signs and Symptoms; Overweight; Depression
• Other Study ID Numbers: AF25

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES

IPD Plan Description

De-identified individual participant data (IPD) underlying the results reported in publications may be shared with qualified researchers for scientific research purposes. Shared data may include demographic, physiological, questionnaire, laboratory, and multi-omics datasets collected as part of the study. The study protocol, statistical analysis plan, informed consent form, and data dictionary may also be made available.

Data will be available beginning 12 months after publication of the primary study results and for up to 10 years thereafter. Access will be granted upon reasonable request, following review and approval of a scientifically sound research proposal by the study investigators and sponsoring institution. Any data sharing will be subject to approval by the responsible ethics committee, where required, and compliance with applicable data protection regulations. Data sharing will further require execution of an appropriate data sharing or transfer agreement to ensure partici

• IPD Sharing Time Frame: 12 months after publication until 10 years after publication
• IPD Sharing Access Criteria: Upon reasonable request and approval
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; SAP

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No