The genioglossus reflex response to sudden onset pulses of negative airway pressure (NAP) in humans is reported to occur more commonly at end rather than onset of expiration when delivered via a mouthpiece. We examined whether this response was modulated by the route of stimulus delivery throughout the respiratory cycle. The genioglossus surface EMG (GGsEMG) response to NAP delivered randomly throughout the respiratory cycle was measured in a set of experiments: (i) 40 stimuli of NAP at -5, -7.5 and -10 cmH2O applied to eight healthy, awake, supine males via nose-mask; and (ii) 60 stimuli of -7.5 cmH2O NAP applied to 15 subjects via both nose-mask and mouthpiece in random order. Despite similar pressure changes being detected in the epiglottis during both routes of stimulus delivery, far lower pressure changes were measured at the nasal choanae during mouthpiece compared with nose-mask delivery. There were no significant differences between the responses during any phase of respiration, nor when NAP was delivered via nose-mask or mouthpiece. We conclude that the sensitivity of the GGsEMG response to NAP in humans does not vary significantly with phase of respiration or route of breathing.