Flow-mediated dilation (FMD) in the brachial artery is a widely used end point in clinical trials despite large within-subject variability and a small dynamic range. Recent studies suggest that the radial artery may be more advantageous for investigating FMD because of an enhanced vasodilator response. This study therefore assessed the validity and repeatability of radial artery FMD (FMD-R) to evaluate its suitability for the noninvasive evaluation of endothelial function. Thirty-three healthy subjects were recruited over a period of 11 months. Intra- and inter-reader reproducibilities were measured with high-resolution ultrasound at 4 time points: twice 1 morning (1 to 3 hours apart) and twice again within 7 days (range 4 to 9 days between visits). Conduit endothelial-dependent and -independent vasomotion were assessed by responses to reactive hyperemia and nitroglycerin, respectively. FMD-R measurements demonstrated significant intra- and interday variabilities (intraclass correlation coefficients [ICCs] 0.38 and 0.23, p = 0.04 and 0.12, respectively). Bland-Altman plots confirmed the test-retest variation in FMD-R. In contrast, radial artery diameter measurements (intra- and inter-reader) demonstrated a high degree of repeatability (interstudy ICC >0.8, p <0.0001). The number of subjects needed to detect a treatment difference of 2% in FMD-R with a p value of 0.05 and a power of 0.80 would be 118 in a crossover design and 234 in a parallel design for assessing group changes. In conclusion, these findings show that FMD-R is highly variable within subjects, even in a healthy population, after adjusting for multiple technical factors and implies biologic variation in radial artery tone.