Background: Physical activity reduces the risk of mortality and age-related chronic diseases, yet its association with biological age measured by DNA methylation (DNAm) clocks remains unclear. This systematic review and meta-analysis aims to evaluate the association between physical activity and biological age measured by DNAm clocks.

Methods: In this systematic review and meta-analysis, we conducted a systematic search of Embase, Cochrane Central Register of Controlled Trials, PubMed, Ovid, Scopus, and Web of Science from Jan 1, 2011, to June 6, 2025, to identify articles on the associations of physical activity and DNAm age, epigenetic age acceleration (EAA), or epigenetic age deviation in humans. Studies were included if they were peer-reviewed, published in English, included a study population with a mean or median age of 18 years or older, and investigated the association between DNAm clocks and physical activity in humans. Studies were excluded if the study population was a disease-specific population without controls. We evaluated risk of bias using an adapted Newcastle-Ottawa Scale and Cochrane Risk of Bias scale. We then performed a random-effects meta-analysis using reported or estimated standardised β coefficients and SEs. We also conducted a publication bias analysis and influence analysis. The study was registered with PROSPERO, CRD42024499021.

Findings: We identified 34 437 articles and, after removal of duplicates and screening, 44 studies were included in the systematic review comprising 145 465 participants: 62 887 (43·2%) females and 82 578 (56·8%) males, with mean ages ranging from 24·1 years to 78·5 years. Across studies, higher levels of physical activity were generally associated with lower DNAm age, although many individual associations did not reach statistical significance. Seven cross-sectional studies contributed to the meta-analysis. Each one SD higher in metabolic equivalent of tasks-min per week was associated with 0·03 SD lower Horvath EAA (β=-0·03 [95% CI -0·05 to -0·01]) and 0·09 SD lower GrimAge EAA (-0·09 [-0·12 to -0·05]). No statistically significant association was observed for Hannum EAA or PhenoAge EAA.

Interpretation: Higher physical activity is significantly associated with lower biological age as measured by Horvath EAA and GrimAge EAA. However, evidence is predominantly from cross-sectional studies, limiting causal inference. Future longitudinal studies and clinical trials using standardised, objectively measured physical activity are warranted to clarify dose-response relationships, and to determine whether physical activity can causally modify ageing trajectories, thereby informing precision strategies for healthy longevity.

Funding: The National University of Singapore and the National Medical Research Council of Singapore.