OBJECTIVE The aim of this study was to evaluate the relationship between retinal blood flow (RBF) and retinal blood oxygen saturation (SO2) in mild to moderate nonproliferative diabetic retinopathy (NPDR) and in age-matched controls. METHODS One eye of each of 15 healthy subjects (68 ± 6 years) and 13 subjects with mild to moderate NPDR (67 ± 10 years) was dilated. None of the patients with NPDR had received treatment for their retinopathic changes or had any evidence of sight-threatening characteristics. Doppler Fourier-domain optical coherence tomography blood flow was measured using the prototype RTVue system; six separate measurements each comprising an upper and a lower nasal pupil scan were acquired. Six hyperspectral retinal measurements were acquired using a noninvasive hyperspectral retinal camera (prototype H-8.5 HR Camera). RESULTS Total RBF was significantly lower in NPDR when compared to controls (42.7 ± 7.5 vs. 33.0 ± 9.2 μL/min; P = 0.004). Mean retinal arterial and venular SO2 were higher in NPDR than in controls (94.7 ± 2.4% vs. 92.9 ± 1.6%, P = 0.02; 62.5 ± 5.7% vs. 56.3 ± 4.7%, P = 0.003). This study showed a correlation between RBF and arteriolar SO2 in both controls (r = 0.58, P = 0.02) and NPDR (r = 0.54, P = 0.05), but no correlation between venular RBF and venular SO2 in controls (r = 0.24, P = 0.83) or in NPDR (r = 0.23, P = 0.45). The arteriovenous difference (AV difference) was lower in the NPDR group when compared to controls (30.6 ± 6 vs. 36.7 ± 5.3, P = 0.008). CONCLUSIONS This study found a lower total RBF and a lower AV difference in the NPDR group, suggesting a reduced oxygen uptake from the retina in people with relatively early diabetic retinopathy.