Each study eye underwent mydriasis and AOSLO imaging in a single-visit study. The instrument's arrangement of four offset aperture images provided two orthogonal split-detector images and enabled isotropic analysis of the arteriolar boundaries. For each arteriole, we calculated the wall-to-lumen ratio (WLR), mean wall thickness, and luminal and external diameters. In total, we enrolled 5 (20.8%) healthy control eyes and 19 eyes of patients with T1D. The DR distribution was: four (16.7%) no-DR, nine (37.5%%) mild or moderate nonproliferative DR (NPDR), and six (25%) severe NPDR or proliferative DR. Mean wall thickness increased significantly in eyes with T1D compared to healthy controls (p = 0.0006) and in eyes with more advanced DR (p = 0.0004). The WLR was significantly higher in eyes with T1D (p = 0.002) or more severe DR (p = 0.004). There was no significant relationship between T1D status or DR severity and any of the arteriolar diameters. In this preliminary study, there appeared to be increases in the WLR and mean wall thickness in eyes with T1D and more severe DR than in the controls and eyes with no/less severe DR. Future studies may further elucidate the relationship between the retinal arteriolar structure and physiologic alterations in DR.