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To evaluate the type and intensity of pain in horses is complicated and there is an ongoing effort to find new ways for an objective pain assessment. Analysis of the heart rate variability (HRV) is a non-invasive method to acquire information about the activity of the autonomous nervous system. It might therefore be useful for pain assessment. The aim of the present study was to evaluate the effects of different intensities and types of pain on HRV and to evaluate, if it can be used for pain assessment. 15 horses with pain were included in the study and served as their own controls, as soon as they were pain-free again. The horses were divided into three disease groups, depending on their underlying disease (disease group 1 = colic, disease group 2 = laminitis, disease group 3 = orthopedic problem). A composite pain scale was used to evaluate the intensity of pain. This pain scale contained a general part, as well as specific criteria for every disease. From a short-term ECG recording, sequences of two minutes length were used for the frequency domain analysis of the HRV. Using a spectral analysis, the low frequency (LF) and high frequency (HF) domain, as well as the LF/HF-ratio were calculated. All parameters measured during pain were compared to controls and in between different pain intensities and disease groups. Horses had low to moderate pain intensities. Regarding the parameters of the HRV, only the LF/HF-ratio was significantly higher in horses with pain (p = 0.028). This is indicative of an increased sympathetic activity because of pain. There were no significant differences between pain intensities or disease groups. A correlation of the HRV with the pain intensity was not evident, either. Therefore the analysis of HRV alone does not seem to be a suitable parameter for pain assessment.One of the main limitations of the present study was the small number of patients. Horses were further given different drugs because of their underlying disease, with unknown effects on the HRV. Further studies with a greater number of horses suffering from pain, without previous medication, would be useful, if this was compatible with the well-being of the animal. Furthermore, standardized protocols for data collection and analysis of HRV should also be established, in order to better evaluate the relevance for pain assessment.