Three-dimensional ultrasound images (3DUS), having two spatial and one temporal dimension, were taken of the brachial artery during baseline conditions, in the transverse and longitudinal planes. The transverse images were analyzed by three different techniques used to quantify flow-mediated dilation (FMD): (1) measuring vessel area manually (TIMA), (2) measuring vessel area semi-automatically (TISA) and (3) measuring vessel diameter (TID). The inter- and intra-observer variability and transducer repositioning variability of each method were compared to each other and to the variability of measurements taken using the traditional method of measuring vessel FMD through measuring vessel diameter on longitudinal images (LID). The percent coefficient-of-variation describing the inter-observer variability (COV(inter)) was similar for the methods, indicating that each method was equally reproducible by the different observers. The percent coefficient-of-variation describing the intra-observer variability (COV(intra)) and the smallest detectable percent change in diameter (Δd(intra)) for each method indicated that TID was the most precise at measuring vessel diameter, and could measure the smallest changes in diameter between successive measurements (COV(intra) = 0.31%, Δd(intra) = 0.87%). LID performed the poorest (COV(intra) = 0.57%, Δd(intra) = 1.59%). The percent coefficient-of-variation describing transducer repositioning (COV(rep)) and the smallest detectable percent change in FMD over time (ΔFMD) for each method indicated that TIMA was the most reproducible method (COV(rep) = 2.35%, ΔFMD = 6.52%) closely followed by TISA. TID performed the poorest (COV(rep) = 5.37%, ΔFMD = 14.89%). TIMA and TISA were found not to be statistically different so we suggest TISA as the method of choice to maximize reproducibility between measurements over time, as it is faster and simpler to perform. In each experiment it was clear that transverse imaging introduced equal or less variability into diameter measurements as compared to longitudinal imaging and we suggest this imaging plane be used in all assessments of FMD.