Highlights
- The first randomised controlled trial comparing two fasting protocols with a comprehensive multi-omics analysis of autophagic flux;
- A novel combination of exercise-induced glycogen depletion with prolonged fasting to enhance autophagy;
- The integration of continuous glucose monitoring and ketone measurements to ensure protocol safety and compliance;
- A systematic investigation of blood-based autophagy biomarkers to move towards easier monitoring in future studies.
Abstract
Importance: Although prolonged fasting has become increasingly popular, the favourable biological adaptations and possible adverse effects in humans have yet to be fully elucidated. Objective: To investigate the effects of a three-day water-only fasting, with or without exercise-induced glycogen depletion, on autophagy activation and the molecular pathways involved in cellular damage accumulation and repair in healthy humans. Design: A randomised, single-centre, two-period, two-sequence crossover trial. The primary outcome is autophagic activity, assessed as flux in peripheral blood mononuclear cells (PBMCs) measured in the context of whole blood. Secondary outcomes include changes in body composition, heart rate variability, endothelial function, and genomic, epigenomic, metabolomic, proteomic, and metagenomic adaptations to fasting in plasma, platelets, urine, stools, and PBMCs. Detailed profiling of circulating immune cell populations and their functional states will be assessed by flow cytometry. Setting: All clinical investigations will be undertaken at the Charles Perkins Centre Royal Prince Alfred Hospital clinic, University of Sydney, Australia. Participants: Twenty-four individuals aged 18 to 70 years, with a BMI of 20–40 kg/m2, free of major health conditions other than obesity. Discussion: While autophagic flux induction through fasting has garnered interest, there is a notable lack of human studies on this topic. This trial aims to provide the most detailed and integrated analysis of how three days of prolonged water-only fasting, combined with glycogen-depleting exercise, affects autophagy activation and other crucial metabolic and molecular pathways linked to cellular, metabolic, and immune health. Insights from this study may pave the way for safe and effective strategies to induce autophagy, offering potential preventive interventions for a range of chronic conditions.