Study Design Randomized controlled trial. Background Spinal manipulation (SM) can trigger a cascade of responses involving multiple systems including the sympathetic nervous system (SNS) and the endocrine system, specifically the hypothalamic-pituitary (HP) axis. However, no manual therapy study has investigated the neuroendocrine response to SM (i.e., SNS-HP axis) in the same trial. Objective To determine short-term changes in SNS activity; heart rate variability (HRV) and endocrine activity (cortisol, testosterone and testosterone/cortisol (T/C) ratio) following a thoracic SM. Methods Twenty four healthy males aged between 18 and 45 years were randomized into two groups: thoracic spinal manipulation (n=12) and sham (n=12). Outcome measures were salivary cortisol (μg/dL), salivary testosterone (pg/mL), T/C ratio, HRV and changes in oxy-haemoglobin (O2Hb) concentration of the right calf muscle (μmol/L). 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<0.05) and salivary cortisol (p<0.01) concentrations. Significant between group differences were noted for salivary cortisol concentration at 5 min (mean difference (MD), 0.35; 95% CI: 0.12to0.6; interaction: p<.001) and T/C ratio at 6 hours post-intervention (MD), -0.91; 95% CI: -1.69to-0.04, p<0.05). However, SM did not differentially alter O2Hb, testosterone or HRV 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, Epub 13 Jul 2017. doi:10.2519/jospt.2017.7348.