The aim was to quantify the magnitude of retinal arteriolar vascular reactivity to a hyperglycemic provocation in diabetic patients stratified by severity of retinopathy and in age-matched controls. The sample comprised 20 non-diabetic controls (Group 1), 19 patients with no clinically visible DR (Group 2), 18 patients with mild-to-moderate non-proliferative DR (Group 3) and 18 patients with diabetic macular edema (Group 4). Retinal hemodynamic measurements using the Canon Laser Blood Flowmeter (CLBF-100) were acquired before and 1 h after drinking a standardized oral glucose load drink. The magnitude of the retinal vascular response, as well as max:min velocity ratio and wall shear rate (WSR), was calculated and compared across groups. A significant change in blood glucose level was observed for all groups (p<0.05). The change in blood glucose elevation was significantly less in Group 1 compared to the other groups. No significant change in arteriolar diameter, blood velocity, blood flow, max:min velocity ratio and WSR was found in patients with diabetes and in age-matched subjects without diabetes. The results of this study indicate that retinal arteriolar blood flow is unaffected by acute elevation of blood glucose using an oral glucose load drink in patients with diabetes and in age-matched controls. This lack of a blood flow response to acute hyperglycemia in patients with early sight-threatening DR may be explained by a loss of retinal vascular reactivity.