BackgroundHeart rate variability (HRV) analysis measures the inter‐beat interval variation of successive cardiac cycles. Measurement of these indices has been used to assess cardiac autonomic modulation and for arrhythmia identification in exercising horses.ObjectivesTo report HRV indices during submaximal exercise, strenuous exercise and recovery, and explore relationships with clinical conditions (arrhythmias, lameness, equine gastric ulcer syndrome [EGUS], lower airway inflammation and upper respiratory tract obstructions [URTOs]) in Thoroughbred racehorses.Study designRetrospective, observational cross‐sectional study.MethodsOne hundred and eighty Thoroughbred horses underwent a treadmill exercise test with simultaneous electrocardiographic recording. Time‐domain HRV indices (standard deviation of the R–R interval [SDRR]; root mean square of successive differences [RMSSD]) were derived for submaximal and strenuous exercise and recovery segments. Clinical conditions (arrhythmia [during each phase of exercise], lameness, EGUS, lower airway inflammation and URTO) were assigned to binary categories for statistical analysis. Relationships between selected HRV indices and the clinical conditions were explored using linear regression models.ResultsDuring submaximal exercise, lameness was associated with decreased logRMSSD (B = −0.19 95% confidence interval [CI] −0.31 to −0.06, p = 0.006) and arrhythmia was associated with increased logRMSSD (B = 0.31 95% CI 0.01–0.608, p = 0.04). During strenuous exercise, arrhythmia was associated with increased HRV indices (logSDRR B = 0.51 95% CI 0.40–0.62, p < 0.001; RMSSD B = 0.60 95% CI 0.49–0.72, p < 0.001). During recovery, arrhythmia was associated with increased HRV indices (logSDRR B = 0.51 95% CI 0.40–0.62, p < 0.001, logRMSSD B = 0.60 95% CI 0.49–0.72, p < 0.001).Main limitationsThe main limitations of this retrospective study were that not every horse had the full range of clinical testing, therefore some horses may have had undetected abnormalities.ConclusionsThe presence of arrhythmia increased HRV in both phases of exercise and recovery. Lameness decreased HRV during submaximal exercise.