Hyperglycemia (HG) is associated with increased mortality and morbidity in acute ischemic events. Regardless of the tissue or organs involved, the vascular endothelium is a key target of ischemia-reperfusion (I/R) injury severity. Among endothelium-protective strategies, exercise has been widely described as useful. However, whether this strategy is able to impact the deleterious effect of HG on endothelial function during I/R has never been challenged. For this, 48 male Wistar rats were randomized into 4 groups: sedentary (Sed) or exercised (Ex, 45 min/day, 5 days/week for 5 weeks) rats, treated (hyperglycemic, HG) or not (normoglycemic, NG) with streptozotocin (40 mg/kg, 48 h before procedure). Vascular I/R (120/15 min) was performed by clamping the femoral artery. Arterial and downstream muscular perfusions were assessed using laser speckle contrast imaging. Vascular endothelial function was assessed in vivo 15 min after reperfusion. HG was responsible for impairment of reperfusion blood flow as well as endothelial function. Interestingly exercise was able to prevent those impairments in the HG group. In agreement with the previous results, HG increased reactive oxygen species production and decreased nitric oxide bioavailability whereas exercise training normalized these parameters. It, therefore, appears that exercise may be an effective prevention strategy against the exacerbation of vascular and muscular damage by hyperglycemia during I/R.