BACKGROUND: Little is known about normal heart rate variability (HRV) in horses during exercise. It can be difficult to separate premature beats from normal beat-to-beat variation at higher heart rates. OBJECTIVES: The aim was to quantify HRV in healthy horses during a high-speed treadmill-standardized exercise test (HSET) and to compare with the HRV in horses observed to have arrhythmias during exercise. ANIMALS: Thirteen healthy horses (Group H), 30 horses with arrhythmias (Group A), and 11 horses with poor performance but no observed arrhythmias (Group O). METHODS: Prospective, observational study. All horses performed a HSET with simultaneous electrocardiograph (ECG) recorded. The ECGs were corrected for artifacts, and arrhythmias noted. Percent instantaneous beat-to-beat cycle length variation (% R-R variation) was calculated, and HRV analyses were performed on trot, canter, and recovery segments. RESULTS: Group H showed between -4.4 and +3.8% R-R variation during trot and between -6.1 and +5.4% R-R variation during the canter phase of the HSET. Group A had significantly larger maximum and 1st percentile R-R shortening and lengthening compared with Group H and Group O during the recovery phase where most arrhythmias were observed. During recovery, a cutoff of 6% maximum % R-R shortening predicted the presence of arrhythmia with 88% sensitivity and 97% specificity and likelihood ratio of 26. CONCLUSIONS AND CLINICAL IMPORTANCE: Healthy horses have little instantaneous R-R variation during exercise. If a cardiac cycle shortens more than 6% from the previous cycle during the recovery phase, this R-R interval is likely to represent an arrhythmic event.