Animal requires a balance between energy intake and energy expenditure to maintain a stable body weight. Exercise which can increase energy expenditure is one of the most important factors affecting energy balance for animals. In order to understand the physiological effects of exercise, changes of body mass, metabolic rate, energy intake, serum leptin and body composition were measured in Eothenomys miletus during 8-weeks of forced exercise training with of treadmill. The results showed that exercise training had no significant effects on body mass, but the metabolic rate and energy intake were increased during 8-weeks exercise training, rest metabolic rate increased 29.9% and locomotor maximal metabolic rate increased 10.7%, respectively. The results also showed that exercise training decrease body fat by 28.9% compared with control group, and exercise training decrease serum leptin concentrations by 27.4% compared with control group. There was a positive relationship between serum leptin concentrations and body fat content in control group, but no for exercise trained group. There were significant increase of the mass of liver and digestive tracts compared with control group, and significant decrease of body water content compared with control group. All results indicated that E.miletus regulates its body mass and energy balance through mobilizing body fat, increasing the metabolic rate and energy intake during long-term forced exercise training. Serum leptin can regulate the variation of body fat during long-term forced exercise training.