BACKGROUND The test dose or hydrolocation technique allows rapid detection of spread location. Though its primary aim is to enhance safety in peripheral nerve blocks, evidence on the potential risks of an intraneural test aliquot is lacking. We conducted a cadaveric study to evaluate the risk of fascicular injury following a low-volume (<1 mL) intraneural injection of the median nerve. METHODS Ten upper limbs from fresh unembalmed human cadavers were studied. In-plane ultrasound-guided intraneural injections of the median nerve were performed at mid, proximal, and distal locations using 1 mL of methylene blue and heparinized blood solution. Nerves were extracted and samples immersed in 10% buffered formalin for 4 weeks. Perpendicular 3 mm slices were obtained for H&E staining and light microscopy analysis. Our main objective was to assess the number of injured fascicles. Secondarily, we evaluated the pattern of intraneural spread. Fascicular injury was defined as the presence perineurium or axonal disruption and/or the presence of erythrocytes inside a nerve fascicle. RESULTS Thirty injections were performed in 10 median nerves. Sonographic swelling was confirmed in 100% of the cases. 352 histological sections were analyzed to assess study outcomes. The mean number of fascicles on each section of median nerve was 20±6 covering 49%±7% of the nerve area. No evidence of axonal disruption nor intra-fascicular erythrocytes was found in any of the analyzed sections. CONCLUSIONS Low-volume intraneural injections do not result in evident fascicular injury. Our findings support the use of a test dose in ultrasound-guided regional anesthesia.