Background: Controlled mechanical ventilation is associated with ventilator-induced diaphragmatic dysfunction, which impedes weaning from mechanical ventilation. To design future clinical trials in humans, a better understanding of the molecular mechanisms using knockout models, which exist only in the mouse, is needed. The aims of this study were to ascertain the feasibility of developing a murine model of ventilator-induced diaphragmatic dysfunction and to determine whether atrophy, sarcolemmal injury, and the main proteolysis systems are activated under these conditions. Methods: Healthy adult male C57/BL6 mice were assigned to three groups: (1) mechanical ventilation with end-expiratory positive pressure of 2-4 cm H2O for 6 h (n = 6), (2) spontaneous breathing with continuous positive airway pressure of 2-4 cm H2O for 6 h (n = 6), and (3) controls with no specific intervention (n = 6). Airway pressure and hemodynamic parameters were monitored. Upon euthanasia, arterial blood gases and isometric contractile properties of the diaphragm and extensor digitorum longus were evaluated. Histology and immunoblotting for the main proteolysis pathways were performed. Results: Hemodynamic parameters and arterial blood gases were comparable between groups and within normal physiologic ranges. Diaphragmatic but not extensor digitorum longus force production declined in the mechanical ventilation group (maximal force decreased by approximately 40%) compared with the control and continuous positive airway pressure groups. No histologic difference was found between groups. In opposition with the calpains, caspase 3 was activated in the mechanical ventilation group. Conclusion: Controlled mechanical ventilation for 6 h in the mouse is associated with significant diaphragmatic but not limb muscle weakness without atrophy or sarcolemmal injury and activates proteolysis.