Physical activity (PA) may delay the onset of age-related diseases by decelerating biological aging. We investigated the association between leisure-time physical activity (LTPA) and metabolomics-based aging markers (MetaboAge and MetaboHealth) in late midlife and during 16 years of follow-up. At the 16-year follow-up, we also investigated the association between device-based PA and MetaboAge and MetaboHealth. We included 1816 individuals (mean age 61.6 years) from the Helsinki Birth Cohort Study at baseline and followed them up for 5 (n = 982) and 16 years (n = 744), respectively. LTPA was assessed via questionnaire at baseline and 16 years later and device-based PA with ActiGraph accelerometer at the 16-year follow-up. Fasting blood samples were applied to calculate MetaboAge acceleration (ΔmetaboAge) and MetaboHealth at baseline and at both follow-ups. Covariate-adjusted multiple regression analyses and linear mixed models were applied to study the associations. A higher volume of LTPA at baseline was associated with a lower MetaboHealth score at the 5-year follow-up (p <
0.0001 for time × LTPA interaction). No associations were detected at the 16-year follow-up. An increase in LTPA over 16 years was associated with a decrease in MetaboHealth score (p <
0.001) and a decrease in LTPA with an increase in MetaboHealth score. Higher device-based PA was associated with a lower MetaboHealth score, but not with ΔmetaboAge. In conclusion, higher LTPA in late midlife and device-based PA in old age were associated with improved MetaboHealth. Increasing LTPA with age may protect against MetaboHealth-based aging. The results support the importance of PA for biological aging in later life.