Summary Background A comprehensive study of the effect of supramaximal exercise in lipid homeostasis of Thoroughbreds provides the basis for future research on the role of lipids on energy metabolism in racehorses. Objective To compare the plasma lipidome of Thoroughbreds before and after supramaximal exercise using an untargeted lipidomics approach. Study design Pilot experimental study. Methods Four Thoroughbred horses were used. The maximal oxygen consumption ( VO 2 max ) was calculated for each horse. Horses then underwent treadmill exercise at the speed for which the oxygen requirements had been calculated to be 115% VO 2 max . Plasma samples were obtained before (T0) and immediately (T1), 15 (T2) and 30 (T3) minutes post‐exercise, and evaluated using liquid chromatography/mass spectrometry. Data analysis consisted of principal component analysis and one‐way repeated measures analysis of variance. Results A total of 933 plasma lipids were detected. Supramaximal exercise‐induced significant changes in the signal intensity of 13 lipids; all ubiquitous in the organism as major components of biological membranes or energy substrates. Main limitations A treadmill was used to replicate track conditions. Also, sample size involved only four horses and the statistical analyses failed to achieve the desired power of 80%. Conclusions The findings in this pilot study suggest that supramaximal exercise induces changes in specific plasma lipids in Thoroughbred racehorses. While the biological significance of these findings remains to be determined, these results provide baseline information for future studies in lipidomics applied to equine exercise physiology. Further research is warranted to better understand the role of lipids on energy metabolism in Thoroughbred racehorses.