Fatigue studies of isolated, intact muscles typically utilize solutions saturated with O2. However, under in vivo fatiguing conditions, less oxygen is delivered to the muscles and they actually experience hypoxia. No studies to date have correlated the effects of acute hypoxia on the isometric contractile properties of intact muscles, skinned fibers isolated from the same muscles, and the cellular content of specific muscle proteins. Therefore, we have studied the effects of in vitro acute hypoxia on the fatigability of intact diaphragm muscle strips and on the isometric contractile properties of single Triton-skinned fibers isolated from control and hypoxic diaphragm muscles. We found that hypoxia and fatiguing stimulation per se affect the tetanic force of intact muscle strips without exhibiting any significant deleterious effects on the calcium-activated force of skinned muscle fibers dissected from the intact muscles. In contrast, fatiguing stimulation under hypoxic conditions decreased both the tetanic force of muscle strips and the calcium-activated force of skinned muscle fibers. Gel electrophoresis of muscles subjected to hypoxia and hypoxic-fatigue revealed that there is a significant reduction in three protein bands when compared to control muscles. Protein modification may be the underlying mechanism of muscle fatigue under physiologic conditions.