The accuracy and reproducibility of a new ambulatory radionuclide detector system (the VEST) for ejection fraction measurement has not been fully validated. Thirty-six subjects, (19 volunteers and 17 patients) underwent repetitive bicycle exercise using sequences of both VEST monitoring and gamma camera imaging. A high intraclass correlation was noted for both absolute ejection fraction [0.84 (0.56, 0.95)] and delta ejection fraction [0.87 (0.63, 0.96)] during repeat VEST monitoring. The intraclass correlation for ejection fraction was comparable for data averaged over 30 sec versus 2 min. These correlations compared favorably to those obtained for assessment of absolute and delta ejection fraction as derived by gamma camera determination by the same computer operator (intraobserver variability), two different computer operators (interobserver variability), and during repeat exercise using gamma camera imaging. In concordance, correlations between VEST and gamma camera measurements were relatively high for both absolute ejection fraction (0.78 [0.61, 0.88]) and delta ejection fraction (0.63 [0.39, 0.79]). Thus, the VEST represents a reproducible means of measuring ejection fraction change during dynamic physical activity. Its accuracy in ejection fraction measurements is similar to gamma camera imaging during exercise testing.