Background: The autonomic nervous system interacts with the pain system. Knowledge on the effects of high velocity low amplitude spinal manipulations (SM) on autonomic activity and experimentally induced pain is limited. In particular, the effects of SM on autonomic activity and pain beyond the immediate post intervention period as well as the relationship between these two outcomes are understudied. Thus, new research is needed to provide further insight on this issue. Objectives: The aim was to assess the effect of a single SM (i.e. SM vs. sham) on cardiovascular autonomic activity. Also, we assessed the relationship between cardiovascular autonomic activity and level of pain threshold after the interventions. Method: We conducted a randomized, cross-over, sham-controlled trial on healthy first-year chiropractic students comprising two experimental sessions separated by 48 h. During each session, subjects received, in a random order, either a thoracic SM or a sham manipulation. Cardiovascular autonomic activity was assessed using heart rate and systolic blood pressure variabilities. Pain sensitivity was assessed using pressure pain threshold. Measurements were performed at baseline and repeated three times (every 12 min) during the post intervention period. Participants and outcome assessors were blinded. The effect of the SM was tested with linear mixed models. The relationship between autonomic outcomes and pressure pain threshold was tested with bivariate correlations. Results: Fifty-one participants were included, forty-one were finally analyzed. We found no statistically significant difference between SM and sham in cardiovascular autonomic activity post intervention. Similarly, we found no post-intervention relationship between cardiovascular autonomic activity and pressure pain threshold. Conclusion: Our results suggest that a single SM of the thoracic spine has no specific effect on cardiovascular autonomic activity. Also, we found no relationship between cardiovascular autonomic activity and pressure pain threshold after the SM. Further experimental research should consider the use of several markers of autonomic activity and a more comprehensive pain assessment.