PURPOSE: This study was conducted to test, in mountain running route conditions, the accuracy of the Polar V800 monitor as a suitable device for monitoring the heart rate variability (HRV) of runners. METHOD: Eighteen healthy subjects ran a route that included a range of running slopes such as those encountered in trail and ultra-trail races. The comparative study of a V800 and a Holter SEER 12 ECG Recorder included the analysis of RR time series and short-term HRV analysis. A correction algorithm was designed to obtain the corrected Polar RR intervals. Six 5-min segments related to different running slopes were considered for each subject. RESULTS: The correlation between corrected V800 RR intervals and Holter RR intervals was very high (r = 0.99, p < 0.001), and the bias was less than 1 ms. The limits of agreement (LoA) obtained for SDNN and RMSSD were (- 0.25 to 0.32 ms) and (- 0.90 to 1.08 ms), respectively. The effect size (ES) obtained in the time domain HRV parameters was considered small (ES < 0.2). Frequency domain HRV parameters did not differ (p > 0.05) and were well correlated (r ≥ 0.96, p < 0.001). CONCLUSION: Narrow limits of agreement, high correlations and small effect size suggest that the Polar V800 is a valid tool for the analysis of heart rate variability in athletes while running high endurance events such as marathon, trail, and ultra-trail races. KEYWORDS: HRV; Open field running conditions; Polar V800 heart rate monitor; Validation