Background-Vascular disease is commonly associated with reduced local synthesis of nitric oxide (NO) and impaired tissue perfusion. We introduce a novel noninvasive, visible-reflectance, hyperspectral imaging technique for quantifying the percentage of hemoglobin existing as oxyhemoglobin (HbO 2 ) as an index of skin tissue perfusion. Methods and Results-To simulate vascular endothelial dysfunction, N G -monomethyl-L-arginine (L-NMMA) was infused into the brachial arteries of 9 healthy subjects for 5 minutes to inhibit forearm NO synthesis, first with the subject breathing room air and subsequently during NO inhalation at 80 ppm for 1 hour. Blood flow was measured by venous occlusion plethysmography, and the percentage of HbO 2 perfusing skin tissue was imaged noninvasively with a visible-reflectance hyperspectral technique. L-NMMA reduced blood flow by 31.7Ϯ4.9% and percentage of HbO 2 by 6.5Ϯ0.1 (Pϭ0.002 and PϽ0.001 versus baseline, respectively). With subjects inhaling NO, blood flow fell during L-NMMA infusion by only 10.9Ϯ7.3%, and the percentage of HbO 2 decreased by 3.6Ϯ0.1 (Pϭ0.007 and PϽ0.001, respectively, versus room air L-NMMA responses). Conclusions-Visible-reflectance hyperspectral imaging demonstrates (1) a significant decline in the percentage of HbO 2 in skin tissue when blood flow is reduced after inhibition of forearm NO synthesis and (2) Key Words: peripheral vascular disease Ⅲ blood flow Ⅲ nitric oxide Ⅲ hemoglobin Ⅲ imaging M ore than 10 million Americans have diabetes mellitus, an important risk factor for cardiovascular disease. 1,2 In view of the morbidity and mortality associated with this condition, methods of early detection of vascular disease are needed to initiate appropriate treatment that might lead to an increased life expectancy and enhanced quality of life. Vascular endothelial dysfunction is common in type I and type II diabetes 3,4 and may result in vasoconstriction because of loss of endogenous synthesis of the vasodilating molecule nitric oxide (NO). 5 This, in turn, may compromise blood flow to the extremities of patients with diabetes and other diseases associated with vascular dysfunction, resulting in reduced tissue oxygenation and potentially leading to ulceration, infection, and loss of limb. An impediment to the appropriate clinical management of patients with peripheral arterial disease is the inability to monitor tissue perfusion noninvasively over time. We introduce a novel, noninvasive, visiblereflectance, hyperspectral imaging technique for assessing vascular endothelial dysfunction and its associated reduction in tissue hemoglobin oxygen saturation.Hemoglobin oxygen saturation may be measured by oximeters, although these devices are somewhat limited in usage. For example, a 2-wavelength transmission device restricts measurements to a single point, by which light is passed through either the finger or earlobe. 6 For application to other parts of the body (as, for example, the chest, forehead, or limbs), a transcutaneous reflectance oximeter was developed and f...