OBJECTIVE: To evaluate the relationship between heart rate variability (HRV) and glycemic status after controlling for metabolic syndrome (MetS) in a healthy working cohort. BACKGROUND: A growing body of literature suggests that reduced HRV, a physiological marker of autonomic nervous system activity, is associated with various pathological conditions including glycemic disorders and cardiovascular diseases. The extent to which this association is confounded by other more traditional markers of cardiovascular risk, such as MetS, is unclear. METHODS: We recruited 2441 study participants (age 17-65) employed at three sites of an airplane manufacturing plant in southern Germany. All subjects underwent medical examination, blood sampling, and 24-hour ambulatory heart rate recording while on their normal work routine. Indices of HRV were determined from readings made throughout the 24-hour examination period, those during either the day or night only as well as a night to day ratio. Pearson correlations and multivariate-adjusted partial correlation coefficients (PCCs) were calculated. RESULTS: Pearson correlations suggested inverse associations between HRV measurements and glycemic status (e.g. High frequency r=-0.07, p<0.001; SDNN r=-0.09, p<0.001). After multivariate adjustment of all other components of MetS (triglyceride, blood pressure, waist circumference, high density lipoproteins), medical and demographic variables, these associations persisted (e.g. High frequency PCC=-0.05, p<0.001; SDNN PCC=-0.06, p<0.001). CONCLUSIONS: We confirm a negative correlation between HRV and glycemic status that appeared to be almost linear in a large cohort of healthy workers. Importantly, we showed that this association was independent of potential confounders, especially all of the MetS components and inflammation.