New Findings: What is the central question of this study? Endothelium-dependent flow-mediated dilatation (FMD) is impaired during acute (60 min) exposure to moderate hypoxia. We examined whether FMD is impaired to the same degree during exposure to milder hypoxia. Additionally, we assessed whether smooth muscle vasodilatory capacity [glyceryl trinitrate (GTN)-induced dilatation] is impaired during acute hypoxic exposure. What is the main finding and its importance? A graded impairment in FMD and GTN-induced dilatation was evident during acute (≤60 min) exposure to mild and moderate hypoxia. This study is the first to document these graded impairments, and provides rationale to examine the relationship between graded increases in sympathetic nerve activity with hypoxia on FMD and GTN-induced dilatation. Endothelium-dependent flow-mediated dilatation (FMD) and endothelium-independent dilatation [induced with glyceryl trinitrate (GTN)] are impaired at high altitude (5050 m), and FMD is impaired after acute exposure (<60 min) to normobaric hypoxia equivalent to ∼5050 m (inspired oxygen fraction ∼0.11). Whether GTN-induced dilatation is impaired acutely and whether FMD is impaired during milder hypoxia are unknown. Therefore, we assessed brachial FMD at baseline and after 30 min of mild (end-tidal PO2 74 ± 2 mmHg) and moderate (end-tidal PET,O2 50 ± 3 mmHg) normobaric hypoxia (n = 12) or normoxia (time-control trial; n = 10). We also assessed GTN-induced dilatation after the hypoxic FMD tests and in normoxia on a separate control day (n = 8). Compared with the normoxic baseline, reductions during mild and moderate hypoxic exposure were evident in FMD (mild versus moderate, −1.2 ± 1.1 versus −3.1 ± 1.7%; P = 0.01) and GTN-induced dilatation (−2.1 ± 1.0 versus −4.2 ± 2.0%; P = 0.01); the declines in FMD and GTN-induced dilatation were greater during moderate hypoxia (P < 0.01). When allometrically corrected for baseline diameter and FMD shear rate under the curve, FMD was attenuated in both conditions (mild versus moderate, 0.6 ± 0.9 versus 0.8 ± 0.7%; P ≤ 0.01). After 30 min of normoxic time control, FMD was reduced (−0.6 ± 0.3%; P = 0.02). In summary, there was a graded impairment in FMD during mild and moderate hypoxic exposure, which appears to be influenced by shear patterns and incremental decline in smooth muscle vasodilator capacity (impaired GTN-induced dilatation). Our findings from the normoxic control study suggest the decline in FMD in acute hypoxia also appears to be influenced by 30 min of supine rest/inactivity.