Simple SummaryStrenuous exercise can increase the chance of exertional heat illness (EHI) in horses. To alleviate or prevent EHI in horses, aggressive human intervention is critical, as well as frequent monitoring of body temperature. If body temperature could be obtained more accurately, safely, and rapidly post-exercise, it could act as a rapid point-of-care in detecting the early stages of EHI in horses. A percutaneous thermal sensing microchip (PTSM) has the potential to provide an accurate body temperature, more rapidly and safely post-exercise. However, to achieve this, the optimal body site for microchip implantation must be determined. Furthermore, PTSMs have only be assessed over a short period of time post-exercise in horses. This study was designed to investigate the relationship between the core body temperature and muscle temperatures measured by PTSMs during exercise, and post-exercise application of different cool-down methods. Identifying the accuracy and usefulness of PTSMs as a means of monitoring the body temperature of horses after strenuous exercise, may help to detect the early stages of EHI, and determine if aggressive human intervention is required.The frequent monitoring of a horse’s body temperature post strenuous exercise is critical to prevent or alleviate exertional heat illness (EHI) from occurring. Percutaneous thermal sensing microchip (PTSM) technology has the potential to be used as a means of monitoring a horse’s body temperature during and post-exercise. However, the accuracy of the temperature readings obtained, and their relationship to core body temperature are dependent on where they are implanted. This study aimed to document the relationship between core body temperature, and temperature readings obtained using PTSM implanted in different muscles, during exercise and post application of different cool-down methods. PTSMs were implanted into the right pectoral, right gluteal, right splenius muscles, and nuchal ligament. The temperatures were monitored during treadmill exercise, and post application of three different cool-down methods: no water application (Wno), water application only (Wonly), and water application following scraping (Wscraping). Central venous temperature (TCV) and PTSM temperatures from each region were obtained to investigate the optimal body site for microchip implantation. In this study, PTSM technology provided a practical, safe, and quick means of measuring body temperature in horses. However, its temperature readings varied depending on the implantation site. All muscle temperature readings exhibited strong relationships with TCV (r = 0.85~0.92, p < 0.05) after treadmill exercise without human intervention (water application), while the nuchal ligament temperature showed poor relationship with TCV. The relationships between TCV and PTSM temperatures became weaker with water application. Overall, however the pectoral muscle temperature measured by PTSM technology had the most constant relationships with TCV and showed the best potential to act as an alternate means of monitoring body temperature in horses for 50 min post-exercise, when there was no human intervention with cold water application.