Changes in arterial shear stress can induce functional and structural adaptations in the vasculature. Studies using in vitro endothelial cell (EC) cultures and isolated vessels from animals have shown that increases in shear stress (similar to what is observed during exercise) can lead to increased expression of total endothelial nitric oxide synthase (eNOS) (1,2) and/or phosphorylated eNOS at serine 1177 (P-eNOSser1177)(3), the primary activation site on eNOS. However, it is unclear whether results obtained in vitro can be extrapolated to ECs in a human artery that is subjected to an increase in blood flow in vivo. Therefore, we aimed to determine if local increases in arterial shear during repetitive muscle contractions induce acute changes in eNOS and P-eNOSser1177 expression in humans. Seven young males (25±1 yr) performed 20 separate bouts (3 min each) of rhythmic forearm exercise at 20% of max over a 2-hr period. Each bout of exercise was separated by 3 min of rest. The switching between exercise and rest allowed subjects to complete the entire protocol without fatiguing and promoted brachial artery shear to remain elevated above baseline throughout the entire 2-hr protocol. An additional six male subjects (24±1 yr) served as time controls (no exercise). ECs were freshly isolated from the brachial artery using sterile J-wires shortly after catheter placement at baseline and again following the 2 hr exercise or time control period. The ECs were incubated with antibodies against Von Willebrand factor and DAPI (to identify ECs with intact nuclei) along with antibodies for eNOS or P-eNOSser1177, followed by fluorescent secondary antibodies to assess protein expression via fluorescence microscopy. Fluorescence intensity for each subject sample was normalized to the intensity of cultured human aortic ECs (HAECs) and expressed as ratios of subject EC protein expression/HAEC. Brachial artery mean shear rate was elevated compared to baseline throughout the course of the 2-hr exercise protocol (Fig. 1, P<0.001). Total eNOS expression did not change in either the exercise (0.13±0.04 vs. 0.12±0.03 a.u.) or time control (0.12±0.03 vs. 0.11±0.03 a.u.) group following each respective trial (P>0.05 for both). However, P-eNOSser1177 was increased in the exercise group (Fig. 2, P=0.02) with no change observed in the time control group (P=0.72). Moreover, there was a moderate yet nonsignificant correlation between the relative changes in mean shear and P-eNOSser1177 in the seven subjects that performed forearm exercise (r=0.65, P=0.11). Our novel results suggest that elevations in brachial artery shear in response to forearm exercise increase eNOSser1177 phosphorylation in ECs of young healthy males. Additionally, our data may provide insight into the beneficial effects of exercise on endothelial function in humans.