Flow-mediated dilatation (FMD) has become a commonly applied approach for assessment of vascular function and health, but methods used to calculate FMD differ between studies. For example, the baseline diameter used as a benchmark is sometimes assessed before cuff inflation, whereas others use the diameter during cuff inflation. Therefore, we compared the brachial artery diameter before and during cuff inflation, and calculated the resulting flow-mediated dilatation (FMD) in healthy children (n=45, 10±1 yrs), adults (n=31, 28±6 yrs) and older subjects (n=22, 58±5 yrs). Brachial artery FMD was examined after 5-minute distal ischemia. Diameter was either determined from 30 seconds before cuff inflation, or from the last 30 seconds during cuff inflation. Edge-detection and wall-tracking of high resolution B-mode arterial ultrasound images was used to calculate conduit artery diameter. Brachial artery diameter during cuff inflation was significantly larger than before inflation in children (P=0.02) and adults (P<0.001), but not in older subjects (P=0.59). Accordingly, FMD values significantly differed in children (11.2±5.1% vs 9.4±5.2%, P=0.02) and adults (7.3±3.2% vs 4.6±3.3%, P<0.001), but not in older subjects (6.3±3.4% vs 6.0±4.2%, P=0.77). When the diameter before cuff inflation was used, an age-dependent decline was evident in FMD, whereas FMD calculated using the diameter during inflation was associated with higher FMD values in older than younger adults. In summary, inflation of the cuff significantly increases brachial artery diameter, which results in a lower FMD response. This effect was found to be age-dependent, which emphasizes the importance of using an appropriate methodology to calculate the FMD.