Time resolved 3D ultrasound (4D ultrasound) is a promising imaging modality to measure geometry and deformation of cardiac structures, because of its high temporal resolution. Previously, we have established 4D ultrasound with speckle tracking to measure the temporally and spatially resolved deformation of the aorta and aortic aneurysms. The measurement accuracy of geometry and deformation measurement by 4D ultrasound was determined by comparison to optical imaging in an in vitro inflation/extension test of porcine aorta, subjected to cyclic loading (five load cases, ranging from pulse pressures of 7 to 90 mmHg and axial prestretches of 1.31 to 1.61). The resulting diameter, diameter change, local displacement and circumferential strain were calculated from 4D ultrasound and optical imaging, respectively. The diameter measurement showed a systematic error of up to –0.55 [0.96] mm (median [IQR]), whereas it was negligible for measures of deformation (up to –0.04 [0.21] mm). The random error of single ultrasound-based measurements was higher for measurements of geometry (95% confidence interval up to ~ 2.5 mm) than for measurements of deformation (95% c.i. up to ~ 0.5 mm). The random error did not scale with the measured deformation amplitude, resulting in relative random errors up to 100% of the measured values for small deformations. The random error could be significantly reduced by averaging of several evaluations of the same ultrasound data set. The reproducibility was assessed by the intraclass correlation coefficient and found to be excellent with values ≥ 0.90. 4D ultrasound-based measurements provide accurate measures of deformation for large blood vessels, with a considerable random error in the case of small deformations, which can be reduced by averaging results from repeated evaluations.