BACKGROUND: Inflammation and vagally mediated heart rate variability (vmHRV) have been implicated in a number of conditions including diabetes and cardiovascular disease. Consistent with the inflammatory reflex termed the \'cholinergic anti-inflammatory pathway\', numerous cross-sectional studies have demonstrated negative associations between vmHRV and inflammatory markers such as C-reactive protein (CRP). The only prospective study, however, showed the opposite: higher CRP at baseline predicted higher high-frequency heart rate variability (HF-HRV) at follow-up. Thus, additional studies are needed to examine the prospective association between vmHRV and CRP. METHODS: Healthy employees participated in a voluntary on-site health assessment. Blood samples and ambulatory heart rate recordings were obtained, and night-time HF-HRV was calculated. Useable heart rate data were available in 2007 for 106 nonsmoking employees (9% women; age 44.4 +/- 8 years), all of whom returned for an identical follow-up health assessment in 2011. Bootstrapped (500 replications) bivariate (r) and partial Pearson\'s correlations (ppc) adjusting for sex, age and body mass index at baseline (2007) were calculated. RESULTS: Zero-order correlations indicated that higher HF-HRV was associated with lower levels of CRP at both time-points (2007: r = -0.19, P < 0.05; 2011: r = -0.34, P < 0.001). After adjustment, HF-HRV remained a significant predictor of CRP (ppc = -0.20, P < 0.05). CONCLUSION: In this study, we have provided in vivo support for the cholinergic anti-inflammatory pathway in humans. Cardiac vagal modulation at baseline predicts level of CRP 4 years later. Our findings have important implications for the role of vmHRV as a risk factor for cardiovascular disease morbidity and mortality. Interventions targeted at vmHRV might be useful in the prevention of diseases associated with elevated systemic inflammation.