BACKGROUND: The study aimed to investigate the link between burnout symptoms and prosocial behaviour, as well as the role of acute stress and vagally-mediated heart rate variability (vmHRV) on this association. METHODS: Seventy men were randomly assigned to either the stress or the control condition of the Trier Social Stress Test for Groups (TSST-G). Prosocial behaviour was assessed via a social decision-making paradigm during the respective TSST-G condition. RESULTS: Correlation analyses revealed negative correlations between prosocial behaviour and burnout symptoms. Acute stress was also associated with reduced prosocial behaviour, whereas no interaction effects with burnout symptoms could be revealed. Exploratory analyses showed that vmHRV was negatively correlated with burnout symptoms during the social decision-making paradigm but did not mediate the link between burnout and prosocial behaviour. CONCLUSION: In conclusion, we report first experimental evidence that burnout symptoms are negatively associated with prosocial behaviour. Further studies are needed to explore the causal relations.