BackgroundThe complex anatomy of peripheral nerves has been traditionally investigated through histological microsections, with inherent limitations. We aimed to compare three-dimensional (3D) reconstructions of median and ulnar nerves acquired with tomographic high-resolution ultrasound (HRUS) and magnetic resonance microscopy (MRM) and assess their capacity to depict intraneural anatomy.MethodsThree fresh-frozen human upper extremity specimens were prepared for HRUS imaging by submersion in a water medium. The median and ulnar nerves were pierced with sutures to improve orientation during imaging. Peripheral nerve 3D HRUS scanning was performed on the mid-upper arm using a broadband linear probe (10–22 MHz) equipped with a tomographic 3D HRUS system. Following excision, nerves were cut into 16-mm segments and loaded into the MRM probe of a 9.4-T system (scanning time 27 h). Fascicle and nerve counting was performed to estimate the nerve volume, fascicle volume, fascicle count, and number of interfascicular connections. HRUS reconstructions employed artificial intelligence-based algorithms, while MRM reconstructions were generated using an open-source imaging software 3D slicer.ResultsCompared to MRM, 3D HRUS underestimated nerve volume by up to 22% and volume of all fascicles by up to 11%. Additionally, 3D HRUS depicted 6–60% fewer fascicles compared to MRM and visualized approximately half as many interfascicular connections.ConclusionMRM demonstrated a more detailed fascicular depiction compared to 3D HRUS, with a greater capacity for visualizing smaller fascicles. While 3D HRUS reconstructions can offer supplementary data in peripheral nerve assessment, their limitations in depicting interfascicular connections and small fascicles within clusters necessitate cautious interpretation.Clinical relevance statementAlthough 3D HRUS reconstructions can offer supplementary data in peripheral nerve assessment, even in intraoperative settings, their limitations in depicting interfascicular branches and small fascicles within clusters require cautious interpretation.Key Points3D HRUS was limited in visualizing nerve interfascicular connections.MRM demonstrated better nerve fascicle depiction than 3D HRUS.MRM depicted more nerve interfascicular connections than 3D HRUS.Graphical