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Vascular response to exogenously administered nitroglycerine, an index of endothelium-independent vasodilation, has been used as a control test for the assessment of endothelium-dependent vasodilation (endothelial function) in humans. However, evidence has been accumulating that not only endothelium-dependent vasodilation but also endothelium-independent vasodilation per se is impaired in individuals with cardiovascular risk factors and cardiovascular disease. Impaired endothelium-independent vasodilation is associated with structural vascular alterations and alterations in vascular smooth muscle cells. Several methods, including assessment of vascular responses to vasoactive agents using angiography in a coronary artery and vascular responses to vasoactive agents using venous occlusion plethysmography and ultrasonography in a peripheral artery, are used to assess endothelium-independent vasodilation in humans. Measurement of endothelium-independent vasodilation is also useful for assessment of atherosclerosis and may be a predictor of future cardiovascular events. In this review, we focus on assessment of endothelium-independent vasodilation from methodology aspects to clinical perspectives.