Background/Aim. Brachial artery flow-mediated dilation (FMD) is extensively used for non-invasive assessment of endothelial function. Traditionally, FMD is calculated as a percent change of arterial diameter from the baseline value at an arbitrary time point after cuff deflation (usually 60 seconds). Considerable individual differences in brachial artery temporal response to hyperemic stimulus have been observed, potentially influenced by the presence of atherosclerotic risk factors (RF). The importance of such differences for the evaluation of endothelial function has not been well established. The aim of the stady was to determine the time course of maximal brachial artery endothelium- dependent dilation in healthy adults with and without RF, to explore the correlation of RF with brachial artery temporal response and to evaluate the importance of individual differences in temporal response for the assessment of endothelial function. Methods. A total of 115 healthy volunteers were included in the study. Out of them, 58 had no RF (26 men, mean age 44 ± 14 years) and 57 had at least one RF (29 men, mean age 45 ± 14 years). High-resolution color Doppler vascular ultrasound was used for brachial artery imaging. To determine maximal arterial diameter after cuff deflation and the time-point of maximal vasodilation off-line sequential measurements were performed every 10 seconds from 0 to 240 seconds after cuff release. True maximal FMD value was calculated as a percent change of the true maximal diameter from the baseline, and compared with FMD value calculated assuming that every participant reached maximal dilation at 60 seconds post cuff deflation (FMD60). Correlation of different RF with brachial artery temporal response was assessed. Results. A maximal brachial artery endothelium-dependent vasodilation occurred from 30-120 seconds after cuff release, and the mean time of endothelium-dependent dilation was 68 ± 20 seconds. Individuals without RF had faster endothelium-dependent dilation (mean time 62 ± 17 seconds), and a shorter timespan (30 to 100 seconds), than participants with RF (mean time 75 ± 21 seconds, time-span 40 to 120 seconds) (p < 0.001). Time when the maximal endotheliumdependent dilation occurred was independently associated with age, serum lipid fractions (total cholesterol, LDL and HDL cholesterol), smoking, physical activity and C-reactive protein. True maximal FMD value in the whole group (6.7 ± 3.0%) was significantly higher (p < 0.001) than FMD60 (5.2 ± 3.5%). The same results were demonstrated for individuals with RF (4.9 ± 1.7% vs 3.1 ± 2.3%, p < 0.001) and without RF (8.4 ± 2.9% vs 7.2 ± 3.2%, p < 0.05). Conclusion. The temporal response of endothelium- dependent dilation is influenced by the presence of coronary FR and individually heterogeneous. When calculated according to the commonly used approach, i.e. 60 seconds after cuff deflation, FMD is significantly lower than the true maximal FMD. The routinely used measurement time-points for FMD assessment may not be adequate for the detection of true peak vasodilation in individual persons. More precise evaluation of endothelial function can be achieved with sequential measurement of arterial diameter after hyperemic stimulus.