The purpose of this study was to determine the physiological and biochemical properties of hindlimb muscles after hypoventilation (HPO) induced by bilateral phrenic nerve denervation. Male Wistar rats (10 weeks-old) underwent HPO by the phrenic nerve denervation at the cervical level or sham surgery. Analyses were performed 4, 8, and 12 weeks after the surgery. The myosin heavy chain (MHC) isoform profile and in vitro isometric contractile properties of the soleus (SOL) and extensor digitrum longus muscles (EDL) were analyzed. From the postoperative period, HPO induced characteristic changes in SpO 2 such as hypoventilation disorder. After 12 weeks, significant increases in MHC1 and significant decreases in MHC2a were observed in the MHC isoform composition in SOL. Moreover, significant increases in MHC2a and significant decreases in MHC2b were also observed in the MHC isoform composition in EDL muscles in the HPO compared with sham (SHM) group. In our study, the tidal volume after unilateral phrenic nerve denervation decreased by approximately 12%, and that after bilateral phrenic nerve denervation decreased by approximately 35%. We concluded that the reduction in behavioral activity levels in the HPO group may have resulted in changes of the peripheral skeletal muscles as a result of disuse atrophy.