Objective: The purpose of the present study was to determine the neuroendocrine response after a thoracic spinal manipulation in people with Achilles tendinopathy. Methods: This was a randomized 2-sequence, 2-period crossover trial. A total of 24 participants, mean (standard deviation) age of 48 (7) years, with a diagnosis of Achilles tendinopathy (>3 mo) were randomly assigned into sequence 1 (sham intervention and then thoracic spinal manipulation) or sequence 2 (thoracic spinal manipulation and then sham intervention). The trial was conducted at a university laboratory with a washout period of 1 week. The primary outcome measure was the testosterone/cortisol (T/C) ratio (salivary samples). The secondary outcome measures included heart rate variability (measured with electrocardiography) and total oxygenation index (nmol/L) of calf muscle and Achilles tendon (measured with near-infrared spectroscopy). A 2-way mixed-model analysis of variance was performed. The statistic of interest was the condition by time interaction. Results: A statistically significant condition by time interaction was found for the T/C ratio (mean difference: -0.16; confidence interval: -0.33 to 0.006; interaction: P < .05) and the total oxygenation index (mean difference: 1.35; confidence interval: -1.3 to 4.1; interaction: P < .05) of calf muscle but not for Achilles tendon (P = .6); however, no difference was found for heart rate variability (P = .5). Conclusion: In people with Achilles tendinopathy, thoracic spinal manipulation resulted in immediate increase in the total oxygenation index in the calf muscle followed by an increase in the T/C ratio 6 hours post-intervention.