Introduction In endurance racing, horses are required to complete distances of up to 160 km in a single day. To date, the energy expenditure (EE) of such effort has not been quantified. This study aimed to evaluate EE during a 160 km endurance race from heart rate (HR) monitoring. Methods Three weeks before competing, two endurance horses (H1 and H2) were submitted to a field exercise test to determine their individual VO2-HR relationship. VO2 and HR were recorded simultaneously at trot (13–14 km/h for 10 min), and at canter (18–19 km/h for 10 min, and 22–23 km/h for 5 min), using the equine K4b2, a portable metabolic measurement system. H1 was then enrolled in Monpazier CEI and H2 in Florac CEI. HR was monitored from 2 minutes before the start to the finishing line of each loop. EE of exercise was calculated from individual VO2-HR relationship and caloric equivalent of oxygen. Results H1 and H2 completed the race at an average speed of 17.02 and 14.46 km/h, respectively. Mean HR was 108 ± 8 and 124 ± 24 and peak HR was 169 and 204 for H1 and H2, respectively. The proportion of time spent at HR≥180 was 0 and 2.7% for H1 and H2 respectively. EE of exercise was evaluated at 177 and 240 MJ for H1 and H2 respectively. Conclusions A 160-km endurance race might present a considerable energy cost, at least 5 times the EE of non-working state. Ethical Animal Research The project was conducted under license from the Department of Health and Animal Care of the French Veterinary Authority (no. A21002) and ethics committee approval n° B0710. Sources of funding: Burgundy Region and AgroSup Dijon. Competing interests: none.