Mules and donkeys are the 5th largest equine population in the U.S., yet basic information such as heart rate and arrhythmia prevalence in mules has not been described. Analysis of heart rate variability (HRV) provides valuable insight into physiologic and autonomic differences between mules, horses, and donkeys. We aimed to pilot the evaluation of heart rate and variability in a healthy population of mules using a 24 h electrocardiogram. Twenty-four-hour ambulatory electrocardiogram (aECG) recordings were collected from an athletic population of seven privately owned healthy mules in Northern California. Vision 5 Holter Analysis Software was utilized for ECG review and analysis. An average heart rate of 32–42 bpm, with a mean of 36 ± 3 bpm for 23.8 ± 0.2 h, was recorded. Minimum heart rate was 16–24 bpm, and maximum was 70–156 bpm. Individual mules had supraventricular complexes, sinoatrial heart block, and second-degree atrioventricular block during the observation period. Electrocardiographic evaluation of mules over 24 h found heart rate (HR) to be similar to horses and lower than in donkeys. Arrhythmias were consistent with the range of cardiac rhythms observed in horses and more frequent than reported in donkeys. Further investigation with a larger sample size of mules is warranted to determine if reference values for horses should continue to be used to assess mules and to investigate the prevalence of arrhythmias and their clinical relevance in the mule.