Study Design Controlled laboratory study. Background Spinal manipulation (SM) can trigger a cascade of responses involving multiple systems, including the sympathetic nervous system and the endocrine system, specifically, the hypothalamic-pituitary axis. However, no manual therapy study has investigated the neuroendocrine response to SM (ie, sympathetic nervous system-hypothalamic-pituitary axis) in the same trial. Objective To determine short-term changes in sympathetic nervous system activity, heart rate variability, and endocrine activity (cortisol, testosterone, and testosterone-cortisol [T/C] ratio) following a thoracic SM. Methods Twenty-four healthy men aged between 18 and 45 years were randomized into 2 groups: thoracic SM (n = 12) and sham (n = 12). Outcome measures were salivary cortisol (micrograms per deciliter), salivary testosterone (picograms per milliliter), T/C ratio, heart rate variability, and changes in oxyhemoglobin concentration of the right calf muscle (micromoles per liter). Measurements were done before and at 5 minutes, 30 minutes, and approximately 6 hours after intervention. Results A statistically significant group-by-time interaction was noted for T/C ratio (P<.05) and salivary cortisol (P<.01) concentrations. Significant between-group differences were noted for salivary cortisol concentration at 5 minutes (mean difference, 0.35; 95% confidence interval: 0.12, 0.6; interaction: P<.01) and for T/C ratio at 6 hours postintervention (mean difference, -0.09; 95% confidence interval: -0.16, -0.04; P = .02). However, SM did not differentially alter oxyhemoglobin, testosterone, or heart rate variability relative to responses in the sham group. Conclusion Thoracic SM resulted in an immediate decrease in salivary cortisol concentration and reduced T/C ratio 6 hours after intervention. A pattern of immediate sympathetic excitation was also observed in the SM group. J Orthop Sports Phys Ther 2017;47(9):617-627. Epub 13 Jul 2017. doi:10.2519/jospt.2017.7348.