Flow-mediated dilatation (FMD) is widely used to describe conduit artery endothelial function. The traditional approaches to FMD calculation assess diameter change at arbitrary time points after occluding cuff deflation. The aim of this study was to examine the time course of brachial artery FMD after a 5-minute period of forearm ischemia in 12 young, 12 fitness matched older and 12 older untrained subjects. Edge-detection and wall tracking of high resolution B-mode arterial ultrasound images, combined with synchronized Doppler waveform envelope analysis, were used to calculate brachial artery diameter, blood flow, and shear rate continuously across the cardiac cycle after forearm ischemia. FMD was significantly higher in young healthy subjects (7.8+/-3.2%) compared with sedentary older subjects (5.2+/-2.8%, P<0.05) but not trained older subjects (6.4+/-2.3%). Time to peak diameter differed between young (50+/-11 seconds) and both older groups (trained; 80+/-21, P<0.001; sedentary: 83+/-36 seconds, P<0.001). A large proportion (>42%) of true peak diameters fell outside the time frames typically used to assess FMD in the literature. When calculated according to the commonly used approach, ie, 60 s after cuff deflation, FMD was significantly lower compared with true peak FMD in all groups (P<0.001), and no differences were evident between the groups. The time course of FMD differs significantly between young and older subjects. Studies assuming that peak dilation occurs at an arbitrary time point, or within limited time windows, may draw misleading conclusions regarding differences between groups. More sophisticated approaches to measurement of FMD are required if it is to be considered a valid biomarker of vascular disease.