bioRxiv2026
BackgroundEpigenetic 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, epigenetic clocks differ substantially in their construction and in the biological signals they capture, and their sensitivity to physiological perturbation may not be a flaw but a consequence of the construction. To understand this more clearly, we undertook a focused investigation of biological reliability for a single, well-characterised perturbation, an overnight fast followed by acute refeeding, examining how and why clock estimates may shift with physiological state. MethodsWe 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. ResultsFasting 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 clocks 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 R2 = 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. ConclusionsEpigenetic clock responses to an overnight fast followed by acute refeeding 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 framework that summarises a clock by its ICC alone, without identifying the biological source of its within-person variation, can misread structured, perturbation-driven biology as measurement noise. 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.