Rationale: Diaphragm weakness in critically ill patients prolongs ventilator dependency and duration of hospital stay and increases mortality and healthcare costs. Themechanisms underlying diaphragm weakness include cross-sectional fiber atrophy and contractile protein dysfunction, but whether additional mechanisms are at play is unknown. Objectives: To test the hypothesis that mechanical ventilation with positive end-expiratory pressure (PEEP) induces longitudinal atrophy by displacing the diaphragm in the caudal direction and reducing the length of fibers. Methods: We studied structure and function of diaphragm fibers of mechanically ventilated critically ill patients and mechanically ventilated rats with normal and increased titin compliance. Measurements and Main Results: PEEP causes a caudal movement of the diaphragm, both in critically ill patients and in rats, and this caudal movement reduces fiber length. Diaphragm fibers of 18-hour mechanically ventilated rats (PEEP of 2.5 cm H-2 O) adapt to the reduced length by absorbing serially linked sarcomeres, the smallest contractile units in muscle (i. e., longitudinal atrophy). Increasing the compliance of titin molecules reduces longitudinal atrophy. Conclusions: Mechanical ventilation with PEEP results in longitudinal atrophy of diaphragm fibers, a response that is modulated by the elasticity of the giant sarcomeric protein titin. We postulate that longitudinal atrophy, in concert with the aforementioned cross-sectional atrophy, hampers spontaneous breathing trials in critically ill patients: during these efforts, end-expiratory lung volume is reduced, and the shortened diaphragm fibers are stretched to excessive sarcomere lengths. At these lengths, muscle fibers generate less force, and diaphragm weakness ensues.