OBJECTIVE: Previous research indicates a link between burnout symptoms and reduced vagally mediated heart rate variability (HRV); however, the directionality of this relationship is still largely unknown. The objective of the present study was to examine the longitudinal relationship between HRV and burnout symptoms for 1 year, with a special focus on the emotional exhaustion (EE) burnout subdimension, which remains inadequately distinguished from overlapping with depressive symptoms. METHODS: Here we present HRV and behavioral data from 167 individuals (mean [SD] age = 43.43 [11.78] years; 30.5% male) who attended two biomarker samplings (T1 and T2) of the Dresden Burnout Study approximately 12 months apart. RESULTS: In hierarchical linear regression analyses, T1 HRV significantly inversely predicted T2 overall burnout symptoms (β = -.16; p = .03) and EE (β = -.23; p = .02), adjusting for age, sex, body mass index, adverse health behaviors, and depressive symptoms. Importantly, only high EE at T1 (β = -.22; p = .04), and not the T1 Maslach Burnout Inventor total score, predicted reductions in HRV from T1 to T2. CONCLUSIONS: We report for the first time longitudinal evidence that HRV is associated with changes in burnout symptoms, independently of depressive symptoms. Results suggest vagal dysfunction being predictive and specific for burnout symptoms, making HRV a promising starting point for the explanation of biophysiological mechanisms underlying burnout symptoms and cardiovascular diseases. The finding of only EE at T1 being predictive for changes in HRV underscores the importance of exhaustion for modulations in autonomic regulation.