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Euthanasia is one of the greatest responsibilities for veterinarian surgeons and should be done with the least possible stress for the patient and its owner. In this prospective study serum cortisol and heartrate variabilities were measured during euthanasia in 40 horses to assess stress levels. Euthanasia was performed by sedating the horses with xylazine (0.8 mg/kg), inducing anesthesia using ketamine (2.2 mg/kg) and diazepam (0.02 mg/kg) and euthanizing the horse using pentobarbital (100 mg/kg). Cortisol levels were measured bevor sedation and after time of death. An ECG was performed during the process. Heartrate variabilities were assessed using Kubios* software. Cortisol levels significantly increased (P = 0.001; Wilcoxon-Test) in all horses between sedation and time of death. Analyzing the data using different groups (horses with colic, orthopedic problems or other diseases; presence or absence of the owner; acute or chronic diseases, and location of euthanasia) there was a smaller increase of cortisol levels when owners were present (P = 0.04; Mann-Whitney test). Furthermore there were significant higher values at both measurements of serum cortisol in horses suffering from colic than in all other horses (before P = 0.01, after P = 0.01; Mann-Whitney test). There was no significant difference whether the horse was euthanized in the stable or in the surgical induction area. Heartrate variabilities showed a significant difference (P < 0.001; Friedman test) during three phases of euthanasia. It was higher during induction, lower during anesthesia and highest during injection of pentobarbital. These results indicate a stress response to the process of euthanasia especially at the time of injecting entobarbital. *Kubios Programm HRV Version 2.0, der Biosignal & Medical Imaging Group, Department f€ur Physik, Universit€at Kuopio, Finland.