Background: Although different types of neural mobilization (NM) exercises induce different amounts of longitudinal nerve excursion and strain, the question whether the increased longitudinal stress and nerve excursion from sliding or tensioning intervention may subtly affect the neural functions has not been answered yet. Objective: To compare the effects of tensioning NM versus sliding NM of the median nerve on peripheral and autonomic nervous system function. Methods: In this randomized controlled trial, 90 participants were randomly assigned to tensioning NM, sliding NM, or sham NM. The neurophysiological outcome measures included peak-to-peak amplitude of the dermatomal somatosensory evoked potential (DSSEP) for dermatomes C6, C7, C8, and T1. Secondary outcome measures included amplitude and latency of skin sympathetic response. All outcome measures were assessed pretreatment, immediately after the two weeks of treatment and one week after the last session of the treatment. Results: A 2-way repeated measures ANOVA revealed significant differences between the three groups. The post hoc analysis indicated that tensioning NM significantly decreased the dermatomal amplitude for C6, C7, C8, and T1 ([Formula: see text]). Sympathetic skin responses in the gliding NM group showed lower amplitudes and prolonged latencies post-treatment when compared to tensioning NM group ([Formula: see text]). In contrast, no significant changes were observed in the DSSEPs and skin sympathetic responses for participants in the sham treatment group ([Formula: see text]). Conclusions: A tensioning NM on the median nerve had a possible adverse effect on the neurophysiology variables of the nerves involved in the neural mobilization. Thus, tensioning NM with the current parameters that place increased stress and strain on the peripheral nervous system should be avoided.