The purpose of this work is to analyse changes in ECG depolarization and repolarization indices and heart rate variability metrics in athletes during 10 km running in mountain route conditions. Eighteen healthy subjects ran a route that included a range of running slopes such as those encountered in trail races. The athletes carried a Holter SEER 12 ECG Recorder™ recording 12-lead ECGs during the race. The ECG signals were resampled from 128 Hz to 1000 Hz. QRS duration, QRS area, R-peak, ST elevation at J-point and J+60 ms, ST slope and T-wave peak indices were computed after signal-averaging the ECG at different stage of running. Also, RR time series during the stages were calculated and short-term heart rate variability (HRV) analysis was done: mean RR interval, the standard deviation of NN intervals (SDNN), the root mean square of differences (RMSSD) of successive NN intervals, power in low frequency (LF; 0.04–0.15 Hz), high frequency (HF; 0.15–0.40 Hz) bands, normalized LF (LFn) and HF (HFn) power and LF/HF ratio. A control before running and six 5-min segments related to different running slopes were considered for each subject: 0%, from 4.5% to 10%, 12%, -12%, from -10% to -4.5% and 0% slopes during mountain route. QRS area and R-Peak were reduced during upslope in leads II and III, comparing to downslope and repose before running. ST slope was significantly higher during upslope stages in leads II, V4 and V5. T-wave amplitude increased significantly in precordial leads during upload running. ST segment depressed in leads II, III and V5 respect control. According to Kruskal Wallis test, mean RR, SDNN, LF and LFn showed high significant differences (p<0.01) among stages and HF and LF/HF were also changing (p<0.05). Changes of ECG and HRV indices can help understanding the cardiac function in runners performing extreme stress.