Insulin resistance is a risk factor for atherosclerosis, and is associated with hyperinsulinemia, abnormal lipid profile and hypertension. Whether hyperinsulinemia affects vascular function independent of insulin resistance or other metabolic risk factors is unknown. This investigation aimed to assess the effects of hyperinsulinemia on endothelial function in subjects with a spectrum of insulin sensitivity and lipid profile. Endothelium-dependent (flow-mediated dilation, FMD) and -independent (nitroglycerin) responses of the brachial artery were studied by high-resolution ultrasound before and during hyperinsulinemia (euglycemic clamp) in 25 normoglycemic, normotensive subjects. Participants were divided in an insulin sensitive and an insulin resistant subgroup based on their sensitivity index (SI_Clamp) values, with a cutoff of 8, and in a normal cholesterol and a high cholesterol subgroup based on their total cholesterol levels, with a cutoff of 5.2 mmol/L (200 mg/dL). In the whole population, FMD was lower during hyperinsulinemia compared to baseline (2.3±0.6% vs 6±0.6%, respectively; P<0.001). Resting FMD was lower in the insulin resistant compared to the insulin sensitive subgroup (4.2±0.9% vs 7.4±0.8%, respectively; P=0.014), and in the high cholesterol compared to the normal cholesterol subjects (4.4±0.7% vs 8±0.7%, respectively; P=0.002). Hyperinsulinemia decreased FMD in both the insulin sensitive (from 7.4±0.8% to 3.6±0.4%; P<0.001) and the insulin resistant subgroup (from 4.2% to 1.22%; P=0.012), and in both the normal cholesterol (from 8±0.7% to 3.9±0.4%; P<0.001) and high cholesterol (from 4.4±0.7% to 1.1±0.8%; P=0.01) participants. Acute hyperinsulinemia impairs conduit vessel endothelial function independent of insulin sensitivity and lipid profile. Insulin may trigger endothelial dysfunction and promote atherosclerosis.