Objective Diffusion tensor imaging (DTI) is a useful approach for studying neuronal integrity in animals. However, the test–retest reproducibility of DTI techniques in animals has not been discussed. Therefore, the first part of this work was to systematically elucidate the reliability of DTI-derived parameters in an animal study. Subsequently, we applied the DTI approach to an animal model of diabetes in a longitudinal manner. Materials and methods In Study 1, nine rats underwent two DTI sessions using the same scanner and protocols, with a gap of 4 weeks. The reliability of the DTI-derived parameters was evaluated in terms of sessions and raters. In Study 2, nine rats received a single intraperitoneal injection of 70 mg/kg streptozotocin (STZ) to develop diabetes. Longitudinal DTI scans were used to assess brain alterations before and 4 weeks after STZ administration. Results In the test–retest evaluation, the inter-scan coefficient of variation (CoV) ranged from 3.04 to 3.73% and 2.12–2.59% for fractional anisotropy (FA) and mean diffusivity (MD), respectively, in different brain regions, suggesting excellent reproducibility. Moreover, rater-dependence had minimal effects on FA and MD quantification, with all inter-rater CoV values less than 4%. Following the onset of diabetes, FA in striatum and cortex were noted to be significantly lower relative to the period where they had not developed diabetes (both P  < 0.05). However, when compared to the control group, a significant change in FA caused by diabetes was detected only in the striatum ( P  < 0.05), but not in the cortex. Conclusion These results demonstrate good inter-rater and inter-scan reliability of DTI in animal studies, and the longitudinal setting has a beneficial effect on detecting small changes in the brain due to diseases.