Several studies have indicated that diaphragm dysfunction develops in patients on mechanical ventilation (MV). Here, we tested the hypothesis that the contractility of sarcomeres, i.e., the smallest contractile unit in muscle, is affected in humans on MV. To this end, we compared diaphragm muscle fibers of nine brain-dead organ donors (cases) that had been on MV for 26 ± 5 h with diaphragm muscle fibers from nine patients (controls) undergoing surgery for lung cancer that had been on MV for less than 2 h. In each diaphragm specimen we determined 1) muscle fiber cross-sectional area in cryosections by immunohistochemical methods and 2) the contractile performance of permeabilized single muscle fibers by means of maximum specific force, kinetics of cross-bridge cycling by rate of tension redevelopment, myosin heavy chain content and concentration, and calcium sensitivity of force of slow-twitch and fast-twitch muscle fibers. In case subjects, we noted no statistically significant decrease in outcomes compared with controls in slow-twitch or fast-twitch muscle fibers. These observations indicate that 26 h of MV of humans is not invariably associated with changes in the contractile performance of sarcomeres in the diaphragm.