The purpose of this study was to examine how two common methods of continuous hypoxemia impact the activity of intracortical circuits responsible for inhibition and facilitation of motor output, and spinal excitability. Ten participants were exposed to 2 hr of hypoxemia at 0.13 fraction of inspired oxygen (FIO2 clamped protocol) and 80% of peripheral capillary oxygen saturation (SpO2 clamped protocol) using a simulating altitude device on two visits separated by a week. Using transcranial magnetic and peripheral nerve stimulation, unconditioned motor evoked potential (MEP) area, short-interval intracortical inhibition (SICI) and facilitation (ICF), and F-wave persistence and area, were assessed in the first dorsal interosseous muscle before titration, 1 and 2 hr of hypoxic exposure, and at reoxygenation. The clamped protocols resulted in differing reductions in SpO2 by 2 hr (SpO2 clamped protocol: 81.9 ± 1.3%, FIO2 clamped protocol: 90.6 ± 2.5%). Although unconditioned MEP peak to peak amplitude and area did not differ between the protocols, SICI during FIO2 clamping was significantly lower at 2 hr compared to SpO2 clamping (P = 0.011) and baseline (P < 0.001). Whereas ICF was higher throughout the FIO2 clamping compared to SpO2 clamping (P = 0.005). Furthermore, a negative correlation between SICI and SpO2 (rrm = -0.56, P = 0.002) and a positive correlation between ICF and SpO2 (rrm = 0.69, P = 0.001) were determined, where greater reductions in SpO2 correlated with less inhibition and less facilitation of MEP responses. Although F-wave area progressively increased similarly throughout the protocols (P = 0.037), persistence of responses was reduced at 2 hr and reoxygenation (P < 0.01) during the SpO2 clamped protocol compared to the FIO2 clamped protocol. After 2 hr of hypoxic exposure, there is a reduction in the activity of intracortical circuits responsible for inhibiting motor output, as well as excitability of spinal motoneurones. However, these effects can be influenced by other physiological responses to hypoxia (i.e., hyperventilation and hypocapnia).