This study was performed to investigate the effect of exercise prehabilitation (EPH) on passive force, residual force enhancement (RFE), and muscle protein expression in rat skeletal muscle. A total of 24 Sprague-Dawley rats (12 weeks old) were randomly divided into three groups: control (CON, n = 8), hindlimb unloading (HLU, n = 8), and exercise prehabilitation (EPH, n = 8). The HLU group underwent hindlimb unloading for 14 days, and the EPH group exercised for 14 days before HLU. Passive force and RFE were measured in the isolated soleus (SOL) muscle. Muscle protein expression and fiber CSA were analyzed by western blot and immunofluorescence. Body weight (BW) was higher in the CON group than in the HLU or EPH groups. Muscle weight (MW)/BW of SOL was lower in HLU than CON. Muscle fiber and physiological CSA were lower in both HLU and EPH groups than in CON. Peak passive force and passive force normalized to physiological CSA of the SOL were lower in treatment groups than CON. RFE (%) was lower in HLU compared to CON, while EPH had higher RFE than HLU in SOL. Myostatin expression was upregulated by HLU but downregulated by EPH. These results suggest that exercise prehabilitation before surgery can enhance muscle function and suppress myostatin expression. EPH may help preserve muscle function by influencing RFE and regulating muscle protein expression, highlighting the potential of exercise prehabilitation in pre-surgical care and rehabilitation.