This study investigated the NO dependence of radial artery (RA) flow-mediated dilation (FMD) in response to 3 different reactive hyperemia (RH) shear stimulus profiles. Ten healthy males underwent 3 RH trials 1) 5min occlusion (5 trial), 2) 10min occlusion (10 trial), and 3) 10min occlusion with cuff re-inflation at 30s (10-30 trial). Trials were performed during saline infusion and repeated during L-NMMA infusion into the brachial artery. RA blood flow velocity was measured with Doppler ultrasound and B-mode RA images were analyzed using automated edge detection software. Shear rate estimation of shear stress, was calculated as the blood flow velocity/vessel diameter. L-NMMA decreased baseline vascular conductance by 35%. L-NMMA infusion did not affect the peak shear rate stimulus (P=0.681) or the area under the curve (AUC) of shear rate to peak FMD (P=0.088). The AUC was significantly larger in the 10 trial vs. the 10-30 or 5 trial (P<0.001). Although %FMD (% change in diameter) in the 10 trial was larger than that in the 5 trial (P=0.035), there was no significant difference in %FMD between the saline and L-NMMA conditions in any trial: 5 trial: 5.62 ±1.48% vs 5.63 ±1.27%; 10 trial: 9.07 ±1.16% vs 11.22 ±2.21%; 10-30 trial: 6.52 ±1.43% vs. 7.98 ±1.51 for saline and L-NMMA, respectively (P=0.158). We conclude the following: 1) RH following 10min of occlusion results in an enhanced stimulus and %FMD compared to 5min of occlusion. 2) When the occlusion cuff is re-inflated 30s post release of a 10min occlusion it does not result in an enhanced %FMD compared to that which results from RH following 5min of occlusion. 3) The lack of effect of L-NMMA on FMD suggests that NO may not be obligatory for radial artery FMD in response to either 5 or 10 min of occlusion in healthy volunteers.