Overproduction of nitric oxide (NO) with attendant oxidative and nitrosative stress has been implicated in sepsis-induced diaphragm dysfunction. Here we determined the impact of controlled mechanical ventilation (MV) on rat diaphragm sarcolemmal injury, inducible NO synthase (iNOS) expression, and oxidative stress during endotoxemia. At 4 h after injection of endotoxin, impaired sarcolemmal integrity and decreased force production by the diaphragm were observed in spontaneously breathing rats. The use of MV during endotoxemia largely eliminated sarcolemmal damage and significantly improved diaphragm force production. These benefits were not associated with alterations in either iNOS expression or protein carbonyls (marker of oxidation), which remained abnormally elevated in septic diaphragms despite MV. Therefore, we hypothesized that the protection afforded by MV was due to its ability to decrease the level of mechanical stress placed on the sarcolemma, because the latter could be hyperfragile in the setting of increased oxidative stress. Using an in vitro system to independently modulate oxidative and mechanical stresses, we confirmed that these two factors act together in a synergistic fashion to favor sarcolemmal injury. Accordingly, our data suggest that MV protects the diaphragm during sepsis by abrogating an injurious interaction between oxidative and biomechanical stresses imposed on the sarcolemma.