Heart Rate variability (HRV) is the inter-beat variability in heart rate and is moderated by the balance of sympathetic and parasympathetic divisions of the autonomic nervous system. Electrocardiography (ECG) can be utilized to obtain Low frequency (LF) to high frequency (HF) ratios that represent sympathetic to parasympathetic response, respectively and these ratios may be increased in people with chronic pain. Spinal manipulation is often used to manage musculoskeletal disorders such as neck pain. This study assesses the influence of cervical manipulation on HRV using LF/HF ratio. Ten subjects without neck pain formed the control condition and passive head movement (PHM) condition during which their head was flexed, extended and rotated. Ten subjects with subclinical neck pain underwent the same conditions. A separate session was performed for an actual manipulation. LabChart™ software was utilized to collect and analyze five minute pre and post R-R intervals. Repeated measures of ANOVA demonstrated significant interaction effect on HRV (F (1, 18) = 6.841, p = 0.018) following manipulation vs. PHM. Subsequent analysis showed a significant decrease in the ratio during manipulation condition (p = 0.0316), that was not seen in any other conditions, suggesting a significant autonomic nervous system alteration. This study may lead to new techniques to assess the effectiveness of various treatment interventions.