Aim: To study the role ofautophagy in muscle establishment with long term regular exercise.Methods: The female mouse exercise model with wheel running scheduled was 6 m/min, 15 min/time, 3 times/d (8:00, 14:00, and 20:00), 5d/week, for 8 weeks.Mice were treated by 1% aqueous solution oftrehalose as a daily drinking water or intraperitoneal injection of chloroquine (10mg/kg) before training in 5 times a week.The analysis of Western blot, TUNEL, HE staining and transmission electron microscopic examination were used to evaluate the autophagy level and muscle fibers structure.Results: Regular wheel running for 8 weeks increased antioxidant capacity, stimulated the formation of autophagosomes, increased LC3-Ⅱ and lowered P62 levels in muscles.Meanwhile, muscle cell morphology and function of mitochondrial were significantly improved in mouse skeletal muscles.The autophagy inducer trehalose had similar effects on muscle cells as exercise, but it failed to further enhance autophagy in mice with exercise training.Chloroquine blocked autophagy flux and caused abnormal mitochondria to accumulate in skeletal muscles.Exercise partially recovered autophagy flux and reduced the detrimental effects ofchloroquine.Conclusion: Regular endurance exercise maintained autophagy at an appropriate level, which played a positive role in muscle establishment and fitness by removing abnormal mitochondria and reducing apoptosis.