Endothelial function is noninvasively assessed by measuring nitric oxide–dependent increase in radial artery diameter accompanying the elevation in shear stress induced by increasing blood flow through a short-lasting ischemia of the hand. However, shear stress also depends on blood viscosity, whose changes might thus affect nitric oxide increase in a manner that is not properly reflected by blood flow changes. In 12 subjects with hemochromatosis, we measured ultrasonographically radial artery diameter and blood flow responses to a 4-minute ischemia of the hand. This was done also after removing 500 mL of blood (and concomitantly infusing 500 mL of saline), which significantly (P<0.01) reduced hemoglobin concentration and hematocrit. The increase in blood flow induced by the 4-minute ischemia was similar before and after blood removal (+76% and +80%), which, in contrast, markedly attenuated the accompanying increase in radial artery diameter (+25% versus +13%,P<0.01). Thus, in humans, blood viscosity is involved in the endothelial response to an increase in shear stress. This implies that this response may not be accurately assessed and compared by quantifying the stimulus only through an increase in blood flow.