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The association between vascular wall shear stress (WSS) and the local development of atherosclerotic plaque makes estimation of in vivo WSS of considerable interest. Three-dimensional ultrasound (3DUS) combined with computational fluid dynamics (CFD) provides a potentially valuable tool for acquiring subject-specific WSS, but the interoperator and intraoperator variability associated with WSS calculations using this method is not known. Here, the accuracy, reproducibility and operator dependence of 3DUS-based computational fluid dynamics were examined through a phantom and in vivo studies. A carotid phantom was scanned and reconstructed by two operators. In the in vivo study, four operators scanned a healthy subject a total of 11 times, and their scan data were processed by three individuals. The study showed that with some basic training, operators could acquire accurate carotid geometry for flow reconstructions. The variability of measured cross-sectional area and predicted shear stress was 8.17% and 0.193 N/m2 respectively for the in vivo study. It was shown that the variability of the examined parameters was more dependent on the scan operators than the image processing operator. The range of variability of geometrical and flow parameters reported here can be used as a reference for future in vivo studies using the 3DUS-based CFD approach.