The aim of this study was to find out whether persistent leisure-time physical activity, adjusted for genetic liability and childhood experiences, protect against occurrence of specific chronic diseases and all-cause mortality. Study design was a 24-year prospective follow-up after 6-year physical activity discordance in twin pairs. From 5,663 healthy adult twin pairs, 146 pairs (including 29 mozygotic) discordant for both intensity and volume of leisure physical activity at baseline in both 1975 and 1981 were systematically identified. Mortality and occurrence of chronic diseases (diabetes, hypertension, coronary heart disease defined according to reimbursable medication status) were followed for the period 1.1.1983–31.12.2006 for mortality and 1.1.1983–31.12.2004 for diseases. By end of follow-up, 19 inactive and 10 active co-twins had died. In the whole sample, HR of death adjusted for social class was 2.08 (95% CI 1.06–4.09) for inactive vs. active co-twins, the HR being 2.67 (95% CI 1.15–6.20) among DZ pairs with no mortality difference among the smaller number of discordant MZ pairs. The reimbursable medication analyses showed a tendency of higher risk for inactive vs. active co-twins. Among DZ pairs, HR of diabetes medication adjusted for social class was 2.73 (95% CI 0.62–12.00) and HR of hypertension medication was 2.14 (95% CI 0.94–4.89). This study supports the earlier findings that physical activity is associated with reduced mortality. However the difference was seen only in DZ pairs and therefore some residual genetic confounding effects on mortality cannot be excluded.