Na,K-ATPase activity is critical for maintaining electrogenesis and skeletal muscle function. Isolated rat diaphragm muscles were studied after 3-h high-altitude hypobaric hypoxia (HAH, corresponds to an altitude of 6000 m) in a pressure chamber. LPO activation was observed over 24 h after HAH; no significant changes in other markers of oxidative stress and energy metabolism were detected. During this period, stable hyperpolarization of the sarcolemma developed due to increased electrogenic activity of the α2-Na,K-ATPase isoform, while the total muscle level of the α2-subunit protein remained unchanged. Our findings may have practical implications for developing measures to maintain skeletal muscle functions during high-altitude adaptation.