As a part of the multi-disciplinary "SELENA-T"-2015 Bed Rest Study, we investigated the pattern of inspiratory muscles fatigue in 22 healthy male subjects during incremental exercise test to exhaustion before and after 21 days of hypokinesia evoked by bed rest. Hypokinesia consisted of head-down bed rest (HDBR) at a minus 6° angle, simulating microgravity present on orbiting spacecraft, in 10 subjects. The remaining 12 subjects spent the first 5 days of hypokinesia in HDBR position and the subsequent 16 days in head-up bed rest (HUBR) at a plus 9.6° angle, as a presumed analog of lunar gravity that is six times less than Earth's gravity. Maximal inspiratory pressure (MIP) and electromyograms (EMG) of the diaphragm (D), parasternal (PS), sternocleidomastoid (SCM), and scalene (S) muscles served as indices of inspiratory muscle function. Before both HDBR and HUBR, exercise decreased MIP and centroid frequency (fc) of EMG (D, PS, SCM, and S) power spectrum (p < 0.05). After 3 weeks of HDBR, but not HUBR, inspiratory muscles fatigue was more expressed compared with control (p < 0.05). We conclude that HDBR lowers inspiratory muscles resistance to fatigue during high-intensity exercise while HUBR has no such effect. These changes may limit maximal ventilation and may contribute to exercise intolerance observed after prolonged simulated microgravity. The physiological mechanisms of respiratory muscle dysfunction after HDBR consist primarily of postural effects, and are not due only to hypokinesia.