Cortico-diaphragmatic pathway was investigated by means of transcranial magnetic stimulation (TMS), in 14 patients affected by definite amyotrophic lateral sclerosis (ALS) without clinical signs of respiratory impairment. Spirometry, gas analysis, and measurement of static inspiratory and expiratory pressures were performed in all patients. Forced vital capacity, forced expiratory volume at the first and second peak expiratory flow, sniff effort from FRC level (SNIP), maximal inspiratory and expiratory pressure at mouth (MIP/MEP), maximal transdiaphragmatic pressure (Pdimx) were considered. TMS was performed, recording by surface electrodes from hemidiaphragm, bilaterally. Latency of cortical and spinal motor-evoked potentials (Cx-MEP/Sp-MEP) and central motor conduction time (CMCT) were measured. None of the patients showed altered spirometry and gas levels. Seven patients showed decreased Pdimx and eight of MEP values. Four patients showed a delayed Sp-MEP. In one patient the Cx-MEP was abolished while the mean values of both Cx-MEP and CMCT were significantly increased (19.2+/-4.1 ms, P<0.0001; 10.8+/-4.8 ms, P<0.0001). Cx-MEP and CMCT did not show significant correlations with any of the respiratory measures. The patients with prolonged Sp-MEP, showed longer disease duration, lower Norris score, lower Pdimx and MEP values. In conclusion, cortico-diaphragmatic study is a sensitive measure to reveal subclinical diaphragmatic impairment although not correlated to respiratory measures.