Non-invasive assessment of brachial artery flow-mediated dilation using cuff occlusion of the arm above or below the elbow to stimulate flow is emerging as a highly useful technique to examine endothelial vasomotor function in human subjects. In anticipation of a large-scale investigation, an important issue is the acceptability of the technique to participants. The purpose of this study was to determine the level of discomfort associated with the technique and compare it to the commonly used procedure of venipuncture. Flow-mediated dilation was determined using cuff occlusion of the arm above the elbow and a blood sample was obtained by standard venipuncture from 54 subjects. The level of discomfort for each procedure was assessed and compared using a visual analogue scale and was found to be extremely low. When the occlusion cuff was positioned above the elbow, the discomfort was slightly more severe (1.9+/-1.9 cm) than venipuncture (1.0+/-1.3 cm, p = 0.003). In 27 subjects, the effect of cuff position (above or below the elbow) was compared: the below the elbow position was associated with a reduction in the percentage increase in flow (570+/-280% versus 900+/-560%, p = 0.005), flow-mediated dilation (6.8+/-3.8% versus 9.8+/-5.7%, p = 0.008) and discomfort (1.6+/-0.8 versus 3.7+/-2.2 cm, p = 0.008). When the cuff was located below the elbow, the level of discomfort was equivalent to that associated with venipuncture. Thus, non-invasive assessment of flow-mediated brachial artery dilation is well tolerated and appears to be suitable for a large-scale study of endothelial function.