The object of this study was to utilize a novel feed-forward active contour (FFAC) algorithm to find a reproducible technique for analysis of brachial artery reactivity. Flow-mediated dilation (FMD) is an important marker of vascular endothelial function but has not been adopted for widespread clinical use given its technical limitations, including inter-observer variability and differences in technique across clinical sites. We developed a novel FFAC algorithm with the goal of validating a more reliable standard. Forty-six healthy volunteers underwent FMD measurement according to the standard technique. Ultrasound videos lasting 5–10 seconds each were obtained pre-cuff inflation and at minutes 1 through 5 post-cuff deflation in longitudinal and transverse views. Automated segmentation using the FFAC algorithm with initial boundary definition from three different observers was used to analyze the images to measure diameter/cross-sectional area over the cardiac cycle. The %FMD was calculated for average, minimum, and maximum diameters/areas. Using the FFAC algorithm, the population-specific coefficient of variation (CV) at end-diastole was 3.24% for transverse compared to 9.96% for longitudinal measurements; the subject-specific CV was 15.03% compared to 57.41%, respectively. For longitudinal measurements made via the conventional method, the population-specific CV was 4.77% and subject-specific CV was 117.79%. The intraclass correlation coefficient (ICC) for transverse measurements was 0.97 (95% CI: 0.95–0.98) compared to 0.90 (95% CI: 0.84–0.94) for longitudinal measurements with FFAC and 0.72 (95% CI: 0.51–0.84) for conventional measurements. In conclusion, transverse views using the novel FFAC method provide less inter-observer variability than traditional longitudinal views. Improved reproducibility may allow adoption of FMD testing in a clinical setting. The FFAC algorithm is a robust technique that should be evaluated further for its ability to replace the more limited conventional technique for measurement of FMD.