Exercise plays a critical role in maintaining and improving the quantity and quality of skeletal muscle. Resistance exercise is especially important improves muscle strength and increase muscle mass. The recent realization of the importance of animal welfare worldwide has emphasized the need for replacing animal experiments with in vitro cell experiments, thereby necessitating the development of a three-dimensional cellular model. Consequently, by applying the existing microfluidic stretch system, this study was conducted to develop a motion-mimicking system that can perform both morphological analysis through immunofluorescence staining and molecular analysis, such as western blotting and quantitative PCR. The system promoted the fusion of C2C12 myoblasts and increased the myotube diameter. In addition, the stretching system enhanced protein synthesis via the AKT-mTOR-p-70 S6K1 pathway and increased expression of Pgc-1 alpha 4, Gpr56, Igf1, Igf2, Nr4a3, Col1a2, Col3a1, and Col4a1, which are known to be induced by resistance exercise. Moreover, the levels of MHC2B-a glycolytic muscle fiber-were increased by stretching, whereas the levels of MHC1-an oxidative muscle fiber-did not vary. In conclusion, our stretching system mimics resistance exercise more closely than endurance exercise and would facilitate morphological and molecular mechanism analyses of changes induced by exercise.