Hypothesis

Repeated 5‑day fasting‑mimicking diet (FMD) cycles paired with periodic exogenous β‑hydroxybutyrate (BHB) supplementation produce a synergistic increase in mitophagic flux that exceeds the effect of FMD alone, leading to greater mitochondrial turnover, improved resting energy expenditure (REE) regulation, and favorable shifts in longevity‑associated biomarkers.

Mechanistic Rationale

FMD activates AMPK and inhibits mTORC1, initiating ULK1‑dependent autophagy [3]. Ketone bodies, particularly BHB, act as endogenous HDAC inhibitors that promote FoxO3 transcriptional activity, upregulating autophagy genes such as LC3B and BNIP3 [2]. Simultaneously, BHB binds to the receptor GPR109A, stimulating PPARα‑PGC‑1α signaling, which drives mitochondrial biogenesis and primes damaged mitochondria for mitophagy [4]. The convergence of AMPK‑ULK1 activation and FoxO3‑PGC‑1α signaling creates a feed‑forward loop: increased mitophagy clears depolarized mitochondria, reducing ROS, which further sustains AMPK activity. This mechanistic synergy predicts a super‑additive rise in mitophagic flux compared with either intervention alone.

Testable Predictions

1. Mitophagy flux, measured by mt‑Keima flow cytometry in PBMCs, will be significantly higher after combined FMD + BHB than after FMD alone or BHB alone.
2. The increase in mitophagy will correlate with a greater reduction in resting metabolic rate (RMR) and a larger rise in fasting β‑hydroxybutyrate concentrations.
3. Improvements in insulin sensitivity (HOMA‑IR) and IGF‑1 suppression will be proportional to the magnitude of mitophagy induction.
4. If mitophagy is blocked pharmacologically (e.g., with ULK1 inhibitor MRT68921), the synergistic benefits on RMR and metabolic markers will be abolished.

Experimental Design

A randomized, crossover trial with 40 healthy adults will undergo three 4‑week conditions separated by 4‑week washout periods: (a) control diet, (b) 5‑day FMD repeated twice per month, (c) FMD + oral BHB (0.3 g/kg) administered each morning during the FMD days. Primary outcomes: mitophagy flux (mt‑Keima), REE (indirect calorimetry), fasting glucose, insulin, HOMA‑IR, IGF‑1, and BHB levels. Secondary outcomes: lipid panel, inflammatory cytokines (IL‑6, TNF‑α), and subjective wellbeing scores. Statistical analysis will use mixed‑effects models to test interaction effects between FMD and BHB.

Potential Outcomes and Falsifiability

If the combined condition yields a statistically significant (>20 %) increase in mitophagy flux relative to FMD alone, and this increase predicts the observed changes in REE and metabolic markers, the hypothesis is supported. Conversely, if mitophagy flux does not differ between FMD and FMD + BHB, or if blocking mitophagy fails to attenuate metabolic improvements, the hypothesis would be falsified, indicating that ketone‑driven HDAC inhibition does not substantially augment FMD‑induced mitophagy in humans.