Measurement of carotid plaque volume and its progression are important tools for research and patient management. In this study, we investigate the observer variability in the measurement of plaque volume as determined by 3-dimensional (3D) ultrasound (US). We also investigate the effect of interslice distances (ISD) and repeated 3D US scans on measurement variability. Materials and Methods Forty 3D US patient images of plaques (range, 37.43 to 604.1 mm3) were measured by manual planimetry. We applied ANOVA to determine plaque volume measurement variability and reliability. Plaque volumes were measured with 9 ISDs to determine the effect of ISD on measurement variability. Additional plaque volumes were also measured from multiple 3D US scans to investigate repeated scan acquisition variability. Results Intraobserver and interobserver measurement reliabilities were 94% and 93.2%, respectively. Plaque volume measurement variability decreased with increasing plaque volume (range, 27.1% to 2.2%). Measurement precision was constant for ISDs between 1.0 and 3.0 mm, whereas plaque volume measurement variability increased with ISD. Repeated 3D US scan measurements were not different from single-scan measurements (P=0.867). Conclusions The coefficient of variation in the measurement of plaque volume decreased with plaque size. The volumetric change that must be observed to establish with 95% confidence that a plaque has undergone change is approximately 20% to 35% for plaques <100 mm3 and approximately 10% to 20% for plaques >100 mm3. Measurement precision was unchanged for ISDs <3.0 mm, whereas measurement variability increased with ISD. Repeated 3D US scans did not affect plaque volume measurement variability.