Fasting Status and Epigenetic Clock Stability: Implications for Aging Research

Background: Epigenetic clocks are DNA methylation-based biomarkers increasingly used in aging research and clinical trials. A recent assessment of 18 clocks across multiple short-term perturbations concluded that most demonstrate only moderate biological reliability, raising concerns about their translational utility. However, understanding biological variability requires understanding the construction of each clock: different clocks capture distinct biological properties that respond differently to specific perturbations, and pooling reliability metrics across heterogeneous populations and array platforms may obscure the mechanisms driving variability in each case. Methods: We evaluated 24 epigenetic clocks spanning five construction categories - first and second generation classical clocks (eg. Horvath, Hannum, PhenoAge), the PC versions of the classical clocks, SystemsAge organ-system clocks, mortality-trained clocks (GrimAge, PCGrimAge, OMICmAge), pace of aging clocks (DunedinPACE) and the IntrinClock, across three datasets: a within-person paired fasting design (n = 15 pairs), a cross-sectional cohort of fasted vs non-fasted (n = 2,895), and EPICv2custom technical replicates (n = 96 samples from 4 individuals). For each clock, we quantified the acute fasting effect with and without immune cell adjustment, decomposed between-person and within-person variance at successive adjustment levels (Raw, EAA, IAA), and benchmarked biological variability against the technical measurement floor. Results: Fasting followed by acute refeeding was associated with group-level shifts of 0.5-3 years in immune-sensitive clocks, while within-person reliability remained high (Raw clock ICC median ~0.96). These observations are compatible because fasting effects are small relative to the age-driven between-person variance that dominates the ICC denominator. The magnitude of the observed shift varied by clock. PC transformations showed larger effects than their classical counterparts in the paired cohort (PC Hannum -2.03 vs. Hannum -1.37 years; PC PhenoAge > PhenoAge; PC Horvath > Horvath), SystemsAge showed the largest effects (1.15-2.9 years younger when fasted), and mortality-trained clocks (GrimAge V1/V2, OMICmAge) and DunedinPACE showed no detectable acute effect (all FDR p > 0.10). Immune cell adjustment attenuated or eliminated the fasting effects in sensitive clocks (PC Hannum 88% attenuation; SystemsAge Blood 99.7%); no clock retained a significant fasting effect after FDR-corrected immune adjustment in either cohort. Within the cross-sectional cohort, a clock's immune content, which is the fraction of its age-independent variance explained by immune cell composition, was correlated with the degree to which immune adjustment attenuated its fasting effect (r = 0.68, p = 0.003). IntrinClock, designed to exclude immune-variable CpGs, showed no fasting effect in either cohort (immune R-squared = 3.2%), serving as a negative control. Technical replicates confirmed near-perfect measurement reproducibility (median Raw ICC > 0.97), establishing that variance in fasting pairs reflects biology, not noise. Immune-adjusted ICCs behaved differently across clocks in ways consistent with their composition: for clocks where fasting generated within-person variance, immune adjustment removed it and ICC increased (SystemsAge EAA 0.768 to IAA 0.913); for clocks unaffected by fasting, immune adjustment removed between-person structure and ICC fell substantially (OMICmAge 0.922 to 0.160), reflecting the estimation cost of fitting many immune cell predictors to stable residuals. Cross-sectional replication (n = 2,895) confirmed immune cell redistribution at scale. Mortality clocks reached significance cross-sectionally despite resistance to acute fasting. Conclusions: Acute refeeding after an overnight fast elicits small shifts in some epigenetic clocks, which varied systematically by training category in our data. PC-based clocks, which concentrate correlated CpG variance including that associated with immune cell composition, showed the largest shifts; mortality-trained clocks showed no detectable acute effect. A reliability-only framework that reports ICC without also testing for systematic group-level effects can miss the kind of structured biological variation observed here under fasting. ICC is not a fixed property of a clock, it is shaped by the study design, the population heterogeneity, the perturbation, and the adjustment applied. We recommend that clock reliability be assessed on a perturbation-specific, clock-by-clock basis, with variance decomposition at each adjustment level and explicit benchmarking against technical replicates.