It is currently unclear to what extent diurnal variation and exercise effect endothelium-dependent nitric oxide (NO)-mediated vasodilation. Therefore, we measured brachial artery flow-mediated dilation (FMD) in 10 males (mean age = 28 yr, SD = 7), before and after a bout of intermittent cycling at 70% peak oxygen uptake on separate days beginning either at 0800 or 1600. Edge-detection and wall-tracking software was used to measure changes in arterial diameter, while shear rate (SR) was assessed using simultaneously derived blood flow velocity and B-mode diameter data. The FMD data were analyzed before and after normalization for SR with repeated-measures models. Before exercise, mean + or - SD FMD was 7 + or - 3% in the morning compared with 11 + or - 6% in the afternoon (P = 0.01). This diurnal variation persisted after data were normalized for SR, which was found to be unaffected by time of day (P = 0.33). Postexercise SR was higher than at baseline (pre-exercise) (P = 0.01) to a similar extent at both times of day. FMD was unaffected by exercise in the morning (P = 0.96) but decreased by 4 + or - 3% following exercise in the afternoon (P = 0.01) so that postexercise measurements did not differ between times of day. These data indicate that endothelium-dependent FMD is lower in the morning, and this finding was not altered by normalization of FMD for diurnal variation in SR. This infers a reduced function of the intrinsic endothelial NO-vasodilator system in the morning. We also report, for the first time, that a bout of intermittent exercise abolishes this diurnal variation in endothelium-dependent FMD.