Sepsis is an inflammatory condition that causes a severe and rapid loss of body proteins, especially skeletal muscles. The ubiquitin-proteasome system plays a major role in skeletal muscle proteolysis. Understanding the effects of exercise preconditioning on septic-induced ubiquitin-proteasome activation plays a pivotal role in planning rehabilitation strategies for patients who are susceptible for developing cachexia. In this study, we applied mild preconditioning exercises in the form of treadmill running for adult mice for a period of two weeks, before they were injected with lipopolysaccharide to induce sepsis. Our results show that the body weight and cross-sectional area (CSA) of muscle fibers were preserved in the pre-exercised mice. The main finding in our study was that pre-exercised mice maintained a low level of tumor necrosis factor-alpha in the gastrocnemius muscle, which resulted in a down-regulated profile of main atrophic mediators: p38, FOXO3A, and multi-ubiquitin proteins. By these findings, we conclude that a mild program of preconditioning exercises can prevent atrophy and preserve muscle mass in acute sepsis. This provides further evidence to the importance of rehabilitation planning in acute illness.