Purpose: To non-invasively examine the effect of acute hypoxia and inspiratory threshold loading (ITL) on inspiratory muscles [sternocleidomastoid (SCM), scalene (SA) and parasternal (PS)] oxygenation in healthy adults using near-infrared spectroscopy (NIRS). Methods: Twenty healthy adults (12 M/8 F) were randomly assigned to perform two ITL tests while breathing a normoxic or hypoxic (FIO2 = 15 %) gas mixture. NIRS devices were placed over the SCM, PS, SA, and a control muscle, tibialis anterior (TA), to monitor oxygenated (O2Hb), deoxygenated (HHb), total hemoglobin (tHb) and tissue saturation index (TSI). With the nose occluded, subjects breathed normally for 4 min through a mouthpiece that was connected to a weighted threshold loading device. ITL began by adding a 100-g weight to the ITL device. Then, every 2 min 50-g was added until task failure. Vital signs, ECG and ventilatory measures were monitored throughout the protocol. Result: Participants were 31 ± 12 year and had normal spirometry. At task failure, the maximum load and ventilatory parameters did not differ between the hypoxic and normoxic ITL. At hypoxic ITL task failure, SpO2 was significantly lower, and ∆HHb increased more so in SA, SCM and PS than normoxic values. SCM ∆TSI decreased more so during hypoxic compared to normoxic ITL. ∆tHb in the inspiratory muscles (SCM, PS and SA) increased significantly compared to the decrease in TA during both hypoxic and normoxic ITL. Conclusion: The SCM, an accessory inspiratory muscle was the most vulnerable to deoxygenation during incremental loading and this response was accentuated by acute hypoxia.