Objectives Long-standing neck pain (LNP) is a clinical condition frequently encountered in the physical therapy clinic. LNP is a complex, multifactorial condition affecting multiple body systems including the autonomic nervous system (ANS). Traditionally, research on the impact of physical therapy on LNP has focused on self-report measures and pain scales. Heart rate variability (HRV) is an objective measure of the ANS, allowing for quantification of effects of treatment. This systematic review is intended to evaluate if manual therapy acutely affects heart rate variability in adults with long-standing neck pain. Methods Pubmed, Medline, CINAHL, Google Scholar, Web of Science, and Cochrane library were used to retrieve the randomized controlled trials for this review between the years 2010–2021. Search terms included: chronic neck pain, neck pain, cervical pain, manual therapy, mobilization, manipulation, osteopathy, osteopathic or chiropractic. Heart rate variability, HRV, heart rate variation, effects, outcomes, benefits, impacts or effectiveness. Results Of 139 articles located and screened, three full-text articles were selected for full qualitative synthesis, with a combined population of 112 subjects, 91 of which were female, with an average age of 33.7 ± 6.8 years for all subjects. MT techniques in three studies were statistically significant in improving HRV in people with LNP; however, techniques were differed across studies, while one study showed no benefit. The studies were found to be of high quality with PEDro scores ≥6. Conclusions Although no clear cause and effect relationship can be established between improvement in HRV with manual therapy, results supported the use of MT for an acute reduction in HRV. No one particular method of MT has proven superior, MT has been found to produce a statistically significant change in HRV. These HRV changes are consistent with decreased sympathetic tone and subjective pain.