The effect of mechanical ventilation on the diaphragm motor cortex remains unknown. We assessed the effect of mechanical ventilation on diaphragm motor cortex excitability by measuring the costal and crural diaphragm motor-evoked potential (MEP) elicited by single and paired transcranial magnetic stimulation. In six healthy subjects, MEP recruitment curves of the costal and crural diaphragms were assessed at relaxed end expiration during spontaneous breathing [baseline tidal volume (Vt(baseline))] and isocapnic volume cycled ventilation delivered noninvasively (NIV) at three different levels of tidal volume (Vt(baseline), Vt(baseline) + 5 ml/kg liters, and Vt(baseline) + 10 ml/kg liters). The costal and crural diaphragm response to peripheral stimulation of the right phrenic nerve was not reduced by NIV. NIV reduced the costal and crural MEP amplitude during NIV (P < 0.0001) with the maximal reduction at Vt(baseline) + 5 ml/kg. Response to paired TMS showed that NIV (Vt(baseline) + 5 ml/kg) significantly increased the sensitivity of the cortical motoneurons to facilitatory (>9 ms) interstimulus intervals (P = 0.002), suggesting that the diaphragm MEP amplitude depression during NIV is related to neuromechanical inhibition at the level of motor cortex. Our results demonstrate that mechanical ventilation directly inhibits central projections to the diaphragm.