Background: Spinal manipulation (SM) has been hypothesized to influence the autonomic nervous system (ANS). Further, it has been proposed that the effects may vary depending on the segment manipulated. The aim of this systematic review was to synthesize the current level of evidence for SM in influencing the ANS in healthy and/or symptomatic population. Methods: Various databases (n = 8) were searched (inception till May 2023) and 14 trials (n = 618 participants) were included in the review. Two authors independently screened, extracted and assessed the risk of bias in included studies. The data were synthesized using standard mean differences and meta-analysis for the primary outcome measures. The Grading of Recommendations, Assessment, Development and Evaluation (GRADE) was used for assessing the quality of the body of evidence for each outcome of interest. Results: Overall, there was low quality evidence that SM did not influence any measure of ANS including heart rate variability (HRV), oxy-hemoglobin, blood pressure, epinephrine and nor-epinephrine. However, there was low quality evidence that cervical spine manipulation may influence high frequency parameter of HRV, indicating its influence on the parasympathetic nervous system. Conclusion: When compared with control or sham interventions, SM did not alter the ANS. Due to invalid methodologies and the low quality of included studies, findings must be interpreted with great caution. Future studies are needed which employ rigorous data collection processes to verify the true physiological implications of SM on ANS. Keywords: Spinal manipulation; autonomic nervous system; heart rate variability; oxy-hemoglobin; sympathetic nervous system.