Endurance exercise performance has been used as a representative index in experimental animal models in the field of health sciences, exercise physiology, comparative physiology, food function or nutritional physiology. The objective of the present study was to evaluate the effectiveness of Fatmax (the exercise intensity that elicits maximal fat oxidation) as an additional index of endurance exercise performance that can be measured during running at submaximal exercise intensity in mice. We measured both Fatmax and Vo2 peak of trained ICR mice that voluntary exercised for 8 weeks and compared them with a sedentary group of mice at multiple inclinations of 20, 30, 40, and 50° on a treadmill. The Vo2 at Fatmax of the training group was significantly higher than that of the sedentary group at inclinations of 30 and 40° (P < 0.001). The running speed at Fatmax of the training group was significantly higher than that of the sedentary group at inclinations of 20, 30, and 40° (P < 0.05). Blood lactate levels sharply increased in the sedentary group (7.33 ± 2.58 mM) compared to the training group (3.13 ± 1.00 mM, P < 0.01) when running speeds exceeded the Fatmax of sedentary mice. Vo2 at Fatmax significantly correlated to Vo2 peak, running time to fatigue, and lactic acid level during running (P < 0.05) although the reproducibility of Vo2 peak was higher than that of Vo2 at Fatmax. In conclusion, Fatmax can be used as a functional assessment of the endurance exercise performance of mice during submaximal exercise intensity.