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.
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
Allocation
NON_RANDOMIZED
Purpose
BASIC_SCIENCE
Masking
NONE
Enrollment
120
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.
University Hospital Zurich
Zurich, Canton of Zurich, Switzerland
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.
Time frame: Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise (single study day).
PBMC Transcriptome
Genome-wide transcriptomic profiling of PBMCs using RNA sequencing to identify exercise-induced changes in gene expression and cellular signaling pathways.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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).
Time frame: 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.
Time frame: 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).
Time frame: 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).
Time frame: 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).
Time frame: 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).
Time frame: 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).
Time frame: 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).
Time frame: 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.
Time frame: 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.
Time frame: Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.
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