Although age-related deterioration of the cardiac function is a well-studied area of research, the interventions and their molecular pathways have not yet been fully identified. Since physical activity is a powerful preventive measure against cardiac aging, our study compared the effects of long-term voluntary and forced physical activity with a sedentary group, utilizing an aging rat model characterized by mitochondrial dysfunction that contributes to age-related cardiovascular diseases. Four experimental groups were created: (I) young controls (12-week-old); (II) 18-month-old aged sedentary rats; (III) aged group with free access to running wheels for 6 months; (IV) aged rats subjected to forced physical activity for 6 months. At the endpoint of the study, the aged animals were two years old. The aged sedentary rats exhibited increased Tei-index, LA/Ao and E/e' ratios as well as decreased e'/a' ratio and lengthened DecT and IVRT, higher perivascular fibrosis ratio and reduced myocardial PKG, STAT3 and Opa1 protein expression, along with decreased ATP synthase (ATPS) activity in comparison to the young controls. In terms of echocardiographic parameters and perivascular fibrosis, the forced running provided more substantial benefits than the voluntary activity demonstrated by decreased Tei-index, E/e' ratio, increased e'/a' ratio and reduced DecT and IVRT. Forced exercise was strongly associated with elevated myocardial expression of PKG, STAT3 and Opa1 proteins and, moreover, the ATPS activity was restored only in the forced running rats. In conclusion, forced but not voluntary exercise has significant protective effects on age-associated diastolic dysfunction by upregulating PKG-STAT3-Opa1 axis and thereby enhancing ATPS activity.