BACKGROUND: This study examined the effects of a mountain ultra-marathon (MUM) on the activity of the autonomous nervous system through heart rate variability (HRV) monitoring and determined whether this variable related to final performance. METHODS: Heart rate and HRV were measured in eight male amateur runners (aged 37-60 years). Measurements were recorded before and after the event, in resting conditions, as well as continuously throughout the whole MUM. In addition, percentage (%) of heart rate reserve (HR<inf>res</inf>) and partial and total times during the race were analyzed. RESULTS: Average heart rate (HR<inf>avg</inf>) measured at rest was increased after the event (+37%). Standard deviation of successive differences (SDSD) and the square root of the mean squared differences of successive NN intervals (RMSSD) were reduced after the MUM (-56% and -59%, respectively). There was a positive relationship between the frequency-domain index normalized low frequency power (PLFn) measured at rest before the event and race time (0.79) while there was a negative relationship between race time and the difference in HR<inf>avg</inf> before and after the event. In the last half of the event, there was a high correlation (Spearman coefficient of correlation >0.9) between race time and the standard deviation of the NN intervals (SDNN) registered during the race. CONCLUSIONS: Autonomous cardiac regulation can be related to the performance in a mountain ultra-marathon. HRV monitoring could represent a practical tool for the evaluation of the relationship between the autonomous nervous system activity and performance in a mountain ultra-marathon.