Defining the role of genes in the genesis of congenital cardiovascular defects involves comparisons of the diameters of arteries measured in wild-type and genetically engineered mouse embryos. This study aims at evaluating the significance and reproducibility of measurements of the diameters of the great intrathoracic arteries of mouse embryos, as produced under routine conditions, by employing a recently suggested measuring method. Using high-resolution episcopic microscopy, we generated digital volume data of 60 mouse embryos (voxel size 1.07 × 1.07 × 2 µm³) of developmental stage 23 according to Theiler. We randomly split the 60 data sets into two groups of 30 and assigned each group to a diploma student. In addition, an experienced scientist received 12 randomly selected specimens of each group. Independently, the researchers created three-dimensional models of the intrathoracic arteries and identified comparable measurement positions along the ascending aorta, pulmonary trunk and descending aorta. At each position, they defined virtual resections cutting through the volume data perpendicular to the longitudinal axis of the artery. In the virtual resections, the researchers measured the perimeter of the lumen of the artery. The diameter was calculated from the perimeter. Then, we performed statistic comparisons of the diameters measured in micrometres and of the ratio of each measured diameter and the diameter of the ascending aorta. Comparisons of the ratios did not reveal statistically significant differences between the measurements created by the different scientists. We assume that the used measuring protocol is highly robust and produces reproducible and significant results under routine conditions.