Background: Current imaging assessment of peripheral artery disease (PAD) relies on anatomical crosssectional visualizations of the affected arteries. Multispectral optoacoustic tomography (MSOT) is a novel molecular imaging technique that provides direct and label-free visualizations of soft tissue perfusion and oxygenation.Methods: MSOT was prospectively assessed in a pilot trial in healthy volunteers (group n 1 =4, mean age 31, 50% male and group n 3 =4, mean age 37.3, 75% male) and patients with intermittent claudication (group n 2 =4, mean age 72, 75% male, PAD stage IIb). We conducted cuff-induced ischemia (group n 1 ) and resting state measurements (groups n 2 and n 3 ) over the calf region. Spatially resolved mapping of oxygenated (HbO 2 ), deoxygenated (Hb) and total (THb) hemoglobin, as well as oxygen saturation (SO 2 ), were measured via hand-held hybrid MSOT-Ultrasound based purely on hemoglobin contrast.Results: Calf measurements in healthy volunteers revealed distinct dynamics for HbO 2 , Hb, THb and SO 2 under cuff-induced ischemia. HbO 2 , THb and SO 2 levels were significantly impaired in PAD patients compared to healthy volunteers (P<0.05 for all parameters). Revascularization led to significant improvements in HbO 2 of the affected limb.Conclusions: Clinical MSOT allows for non-invasive, label-free and real-time imaging of muscle oxygenation in health and disease with implications for diagnostics and therapy assessment in PAD.