Hypothesis

We propose that the magnitude of resting metabolic rate (RMR) reduction induced by prolonged fasting directly reflects the intensity of autophagic flux, and that individuals exhibiting both a large RMR drop (>40 kcal/day beyond weight‑loss expectations) and high autophagic activity (measured as LC3‑II turnover in peripheral blood mononuclear cells) will show superior improvements in healthspan biomarkers after a 6‑month fasting regimen compared with those showing only one of these responses.

Mechanistic rationale

Prolonged fasting activates AMPK and inhibits mTOR, two signaling hubs that concurrently promote mitochondrial uncoupling (lowering RMR) and ULK1‑dependent autophagy initiation【1†L1-L3】【2†L1-L2】. Recent work shows that AMPK‑driven phosphorylation of ACC reduces malonyl‑CoA, relieving inhibition of CPT1 and increasing fatty‑acid oxidation, which uncouples respiration and generates heat, thereby decreasing basal energy expenditure【3†L1-L4】. Simultaneously, AMPK phosphorylates ULK1 at Ser317/Ser777, triggering autophagosome formation【4†L1-L3】. We argue that the degree of AMPK activation determines the balance between these outputs: a strong AMPK signal yields a pronounced RMR reduction coupled with robust LC3‑II lipidation, whereas a weaker signal produces a modest metabolic slowdown with limited autophagy.

Testable predictions

1. Correlation – In a cohort of healthy adults undergoing a 72‑hour fast, the percent change in RMR measured by indirect calorimetry will correlate positively (r > 0.5) with the fold‑increase in LC3‑II after bafilomycin A1 blockade in PBMCs.
2. Stratification – Participants split into high‑RMR‑drop/high‑LC3‑II, high‑RMR‑drop/low‑LC3‑II, low‑RMR‑drop/high‑LC3‑II, and low‑RMR‑drop/low‑LC3‑II groups will differ in 6‑month changes in insulin sensitivity (HOMA‑IR), inflammatory cytokines (IL‑6, TNF‑α), and cardiorespiratory fitness (VO₂max). The high‑RMR‑drop/high‑LC3‑II group will exhibit the greatest improvements.
3. Falsification – If no significant correlation exists between RMR reduction and LC3‑II turnover, or if the high‑RMR‑drop/high‑LC3‑II stratum does not outperform the other strata on healthspan outcomes, the hypothesis is falsified.

Experimental design

• Recruit 120 adults aged 30‑50, BMI 22‑30 kg/m².
• Baseline measurements: RMR (indirect calorimetry, 20‑min resting), autophagy flux (LC3‑II Western blot with/without bafilomycin A1 in isolated PBMCs), HOMA‑IR, plasma IL‑6/TNF‑α, VO₂max.
• Intervention: Three consecutive 72‑hour water‑only fasts spaced two weeks apart (total of three fasts over six weeks).
• Follow‑up: Repeat all measurements at 3 months and 6 months post‑intervention.
• Statistical analysis: Pearson correlation for prediction 1; two‑way ANOVA (RMR stratum × autophagy stratum) for prediction 2; equivalence testing for falsification criteria.

Expected impact

Confirming that metabolic slowing is not merely a side‑effect but a readable biomarker of autophagy potency would enable personalized fasting prescriptions. It would also reconcile contradictory reports where caloric restriction improves healthspan without marked weight loss, suggesting that the metabolic‑autophagy coupling, rather than calorie count per se, drives longevity benefits【5†L1-L4】【6†L1-L3】.

Broader implication

If the link holds, pharmacological AMPK activators that mimic the fasting‑induced RMR drop without nutrient deprivation could be evaluated for their capacity to induce autophagy and improve healthspan, offering an alternative for individuals unable to adhere to prolonged fasts【7†L1-L2】.