Little is known about the structural response of the diaphragm to controlled mechanical ventilation. We examined effects of this intervention on muscle mass, myosin heavy chain isoforms, and contractile function in the rat diaphragm. Animals were mechanically ventilated for up to 4 days, and comparisons were made with normal control rats as well as spontaneously breathing animals anesthetized for the same duration as the mechanical ventilation group. The diaphragm-to-body weight ratio was significantly reduced in the mechanical ventilation group only. After mechanical ventilation, an increase in hybrid fibers coexpressing both type I (slow) and type II (fast) myosin isoforms was found within the diaphragm, which occurred at the expense of the pure type I fiber population. In contrast, the percentages of type I, type II, and hybrid fibers in the limb muscles (soleus and extensor digitorum longus) did not differ between experimental groups. The optimal length for force production, as well as maximal force-generating capacity of the diaphragm, was also significantly decreased in mechanically ventilated animals. We conclude that even short-term controlled mechanical ventilation produces significant remodeling and functional alterations of the diaphragm, which could impede efforts at discontinuing ventilatory support.