Introduction: Increased physical activity (PA) reduces cardiovascular disease (CVD) risk but the molecular mechanisms are not well understood. Previous studies indicate that physical activity (PA) can exert epigenetic effects by altering gene-specific DNA methylation. The aim of the present study was to investigate the relationship between PA and DNA methylation of genes known to be implicated in CVD. Methods: Blood samples were collected at baseline and after 8 years from 253 white females (70.7 ± 0.3 yrs) and 137 white males (71.7 ± 0.4 yrs) in the Cardiovascular Health Study. Leukocyte DNA was bisulphite-modified, and methylation determined by quantitative methylation-specific PCR (QMSP). Anthropometric and self-report physical activity data were collected at both time points and the latter used to calculate physical activity energy expenditure (PAEE; kcal/wk). Participants were stratified by extent of change in PAEE over the 8 years; a decrease by >500 kcal/wk (DEC), no overall change (NOC) and an increase by >500 kcal/wk. N-way ANCOVA with post-hoc multiple t-tests with the Bonferroni correction was used to locate differences. All results are reported as mean ± S.E.M and deemed significant if P < 0.05.Results: A decrease in PAEE of 500 kcal/wk or more resulted in no change in methylation, whereas those who maintained or increased PAEE by 500 kcal/wk or more had significantly increased methylation of TNF (Figure 1a). Similarly, those who increased PAEE by 500 kcal/wk had significantly reduced methylation of IL10 compared with those who decreased PAEE by 500 kcal/wk (Figure 1b). These changes were independent of any effects of ageing or changes in waist circumference.Discussion: Changes in PAEE of more than 500 kcal/wk were significantly associated with the differential methylation of TNF and IL10 in leukocytes of aged subjects. These genes are strongly implicated in CVD risk. Importantly, PAEE was associated with a change in methylation that would presumably benefit cardiovascular health. These results suggest that PA-induced effects on DNA methylation could underpin the reported associations between PA and CVD risk..