AbstractVarious morphological and functional parameters of peripheral nerves and their vascular supply are indicative of pathological changes due to injury or disease. Based on recent improvements in optoacoustic image quality, the ability of multispectral optoacoustic tomography, to investigate the vascular environment and morphology of peripheral nerves is explored in vivo in a pilot study on healthy volunteers in tandem with ultrasound imaging (OPUS). The unique ability of optoacoustic imaging to visualize the vasa nervorum by observing intraneural vessels in healthy nerves is showcased in vivo for the first time. In addition, it is demonstrated that the label‐free spectral optoacoustic contrast of the perfused connective tissue of peripheral nerves can be linked to the endogenous contrast of hemoglobin and collagen. Metrics are introduced to analyze the composition of tissue based on its optoacoustic contrast and show that the high‐resolution spectral contrast reveals specific differences between nervous tissue and reference tissue in the nerve's surrounding. How this showcased extraction of peripheral nerve characteristics using multispectral optoacoustic and ultrasound imaging could offer new insights into the pathophysiology of nerve damage and neuropathies, for example, in the context of diabetes is discussed.