The combined effect of creatine (Cr) or estrogen (E(2)) with exercise training on cardiac reserve function and antioxidant reservation against oxidative stress were investigated in ovariectomized female Golden Syrian hamsters. One hundred animals were divided into nonexercise and exercise-trained groups, in which each group was separated into the control and 4 treatments of Cr depletion (Cr-), Cr supplementation (Cr+), E(2) replacement (E(2)), and Cr supplementation combined with E(2) replacement (Cr+E (2)). In the exercise-trained group, wheel-running exercise (10 minutes a day, 5 days a week) was imposed for 9 weeks. After the animals were sacrificed, several indicators of cardiac function, specifically the corrected QT interval, left ventricular developed pressure (LVDP), and maximum rate of rise (dP/dt(max)) against a hydrogen peroxide (H(2)O(2)) stress test were measured in isolated hearts using the Langendorff apparatus. Markers of oxidative stress, in other words, reduced glutathione (GSH), oxidized glutathione (GSSG), and an antioxidant enzyme, glutathione peroxidase (GPx) were determined. Exercise-trained animals could restore cardiac reserve function and antioxidant levels against oxidative damage (P<0.05). Cr+, E(2) , and Cr+E(2) combined with exercise training showed highly protected cardiac reserve function against oxidative stress compared to Cr+, E(2) , and Cr+E(2) without exercise (P<0.05). The myocardial antioxidant levels were improved greatly in E(2) and Cr+E(2) combined with exercise training (P<0.05). In conclusion, estrogen replacement and creatine supplementation plus estrogen replacement when combined with exercise training show significant protective effects for cardiac reserve function and antioxidant reservation against oxidative stress in estrogen-deficient hamsters.