Label-free multispectral optoacoustic tomography (MSOT) has recently shown superior performance in visualizing the morphology of human vasculature, especially of smaller vessels, compared to ultrasonography. Herein, we extend these observations towards MSOT interrogation of macrovascular endothelial function. We employed a real-time handheld MSOT scanner to assess flow-mediated dilatation (FMD), a technique used to characterize endothelial function. A data processing scheme was developed to quantify the dimensions and diameter changes of arteries in humans and determine wall distensibility parameters. By enabling high-resolution delineation of the blood-vessel wall in a cross-sectional fashion, the findings suggest MSOT as a capable alternative to ultrasonography for clinical FMD measurements.