Aged garlic extract (AGE) has recently received attention as a potent anti-fatigue agent. The principal aim of this study was to elucidate the mechanism responsible for the ameliorating effect of AGE on physical fatigue in rats caused by repeated endurance exercise on a mechanical treadmill apparatus. Rats were subjected to endurance exercise 5 times per week for 4 weeks. AGE at a dosage of 2.86 g/kg was administrated to rats 30 min before every exercise. Succinate dehydrogenase (SDH) activity in the gastrocnemius and soleus muscles and superoxide dismutase (SOD) activity, nitric oxide (NO) metabolites, and lactic acid concentration in plasma were evaluated as biomarkers of physical fatigue. SDH activity was increased 2-4-fold by repeated endurance exercise in comparison with unexercised (intact) rats, and AGE further up-regulated this activity by 40%. SOD activity was increased 5-fold, whereas AGE maintained it at a level equivalent to that in intact rats. Levels of NO metabolites were slightly decreased, whereas AGE enhanced them 2-fold. Lactic acid concentration was not changed in any of the groups. These results indicate that AGE may facilitate the turnover of aerobic glucose metabolism, attenuate oxidative stress, and promote oxygen supply based on vasodilation, suggesting that AGE ameliorates the various impairments associated with physical fatigue.