Consistent, repeatable tumour volume measurements are key to accurately calculating tumour growth and successful assessment of therapeutic efficacy. Our previous work showed that a novel 3D and thermal imaging system for measuring subcutaneous rodent tumours (BioVolume) significantly reduced inter-operator variability across three in vivo efficacy studies. Here we continue to investigate this reduction in inter-operator variability across a much larger dataset. A dataset of 5,593 inter-operator repeats and 5,073 corresponding calliper repeats was obtained from tumour scans and measurements in 22 laboratories across 238 studies with 112 users, 23 animal strains and 98 unique cell lines. The inter-operator variability of the two measurement methods was analysed using coefficient of variation (CoV), intra-class correlation (ICC) analysis, and significance testing. The 3D and thermal imaging system produced a significantly lower median CoV of 0.173 compared to a median calliper CoV of 0.205 (P value = 5.2 x 10^-9). ICC analysis further confirmed the statistical significance of these values, allowing us to conclude that this novel 3D and thermal imaging system offers a significant reduction in inter-operator variability. This has the potential to improve reproducibility of in vivo studies across a wide range of animal strains and cell lines. The effects of using a device with large inter-operator variability at critical points in the study were also investigated. At randomisation, changing the operator performing measurements resulted in 59.4% probability that a rodent would be reassigned to a different group. For measurements carried out using the imaging system, the probability that changing the operator would also result in change of a rodent’s group was much lower at 29.2%. During studies where the tumour was expected to regress, substituting an operator mid-study resulted in a tumour volume increase of approximately 500mm^3 when callipers were used for measurement. For the imaging device, substituting users did not affect the tumour regression trend, potentially removing the need for the same operator to be present for the entire study duration. The effect of swapping an operator mid-study on the drug efficacy metric AUC (Area Under the Curve) was also observed; no statistical difference in AUC was observed for both BioVolume and callipers (overlapping 95% confidence intervals).