The effects of a standard protocol for euthanasia on heart rate variability (HRV) as a consequence of stress response were analyzed in this prospective clinical study. The HRV was determined in 40 horses undergoing euthanasia due to various reasons, at different locations, and with/without owner presence. For euthanasia, horses were sedated with xylazine or a combination of xylazine and butorphanol. General anesthesia was induced using diazepam and ketamine. Afterwards, horses were euthanized with pentobarbital. The ECG data were taken by a Telemetric ECG at three time points (sedation, anesthesia, anesthesia until death). The HRV was analyzed including the low (LF) and high frequency (HF) components of HRV and the sympathovagal balance (LF/HF ratio). Significant differences in the LF, HF and LF/HF ratio were found between the three time points of euthanasia (p < 0.001). The HRV analysis showed dominating sympathetic activity in the preparation phase of euthanasia and during the injection of pentobarbital. The location of euthanasia, presence of owner and type of primary diseases had no influence on stress parameters. Horses showing excitations or groaning during euthanasia did not differ in HRV. Horse with colic were however more likely to show reoccurrence of breathing during euthanasia. In conclusion, HRV is a sensitive, noninvasive parameter to obtain sympathovagal stimulations during euthanasia and adapted protocols for euthanasia in horse with colic should be studied.