OBJECTIVE: Hyperglycemia and autonomic dysfunction are bidirectionally related. We investigated the association of longitudinal evolution of heart rate variability (HRV) with incident type 2 diabetes (T2D) among the general population. METHODS: We included 7630 participants (mean age 63.7 years, 58% women) from the population-based Rotterdam Study who had no history of T2D and atrial fibrillation at baseline and had repeated HRV assessments at baseline and during follow-up. We used joint models to assess the association between longitudinal evolution of heart rate and different HRV metrics (including the heart-rate corrected standard deviation of the normal-to-normal RR intervals (SDNNc), and root mean square of successive RR-interval differences (RMSSDc)) with incident T2D. Models were adjusted for cardiovascular risk factors. Bidirectional Mendelian randomization (MR) using summary-level data was also performed. RESULTS: During a median follow-up of 8.6 years, 871 individuals developed incident T2D. One standard deviation (SD) increase in heart rate (hazard ratio [HR], 1.20, 95% confidence interval (CI), 1.09-1.33), and log(RMSSDc) (1.16, 95% CI 1.01-1.33) were independently associated with incident T2D. The HRs were 1.54 (95% CI 1.08-2.06) for participants younger than 62 years and 1.15 (95% CI 1.01-1.31) for those older than 62 years for heart rate (p for interaction <0.001). Results from bidirectional MR analyses suggested that HRV and T2D were not significantly related to each other. CONCLUSIONS: Autonomic dysfunction preceeds development of T2D, especially among younger individuals, while MR analysis suggests no causal relationship. More studies are needed to further validate our findings.