OBJECTIVE Low heart rate variability (HRV), a marker for cardiac autonomic dysfunction, is a known feature of type 2 diabetes, but it remains incompletely understood whether this also applies to prediabetes or across the whole glycemic spectrum. Therefore, we investigated the association among prediabetes, type 2 diabetes, and measures of glycemia and HRV. RESEARCH DESIGN AND METHODS In the population-based Maastricht Study (n = 2,107; mean ± SD age 59 ± 8 years; 52% men; normal glucose metabolism [n = 1,226], prediabetes [n = 331], and type 2 diabetes [n = 550, oversampled]), we determined 24-h electrocardiogram-derived HRV in time and frequency domains (individual z-scores, based upon seven and six variables, respectively). We used linear regression with adjustments for age, sex, and major cardiovascular risk factors. RESULTS After adjustments, both time and frequency domain HRV were lower in prediabetes and type 2 diabetes as compared with normal glucose metabolism (standardized β [95% CI] for time domain: −0.15 [−0.27; −0.03] and −0.34 [−0.46; −0.22], respectively, P for trend <0.001; for frequency domain: −0.14 [−0.26; −0.02] and −0.31 [−0.43; −0.19], respectively, P for trend <0.001). In addition, 1-SD higher glycated hemoglobin, fasting plasma glucose, and 2-h postload glucose were associated with lower HRV in both domains (time domain: −0.16 [−0.21; −0.12], −0.16 [−0.21; −0.12], and −0.15 [−0.20; −0.10], respectively; frequency domain: −0.14 [−0.19; −0.10], −0.14 [−0.18; −0.09], and −0.13 [−0.18; −0.08], respectively). CONCLUSIONS Both prediabetes and type 2 diabetes were independently associated with lower HRV. This is further substantiated by independent continuous associations between measures of hyperglycemia and lower HRV. These data strongly suggest that cardiac autonomic dysfunction is already present in prediabetes.