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Introduction: Alternatives for out-of-clinic heart rate (HR) measurement are required to optimise the management of atrial fibrillation (AF) in dogs. Additionally, the presence of circadian variation (CV) in HR in pet dogs remains unknown. We aimed to identify the number and duration of spot-checks required for an accurate estimation of 24-hour HR in canine AF. Circadian variation in HR was examined in healthy dogs and dogs with AF, and spot-check-derived HR was compared with a CV-derived gold standard., Materials and Methods: Ambulatory electrocardiogram data from healthy dogs and dogs with AF were retrospectively analysed. Heart rate was calculated from the entire recording and pre-defined periods (spot-checks) of one hour to 30 and 60 s in duration. Circadian variation in HR was determined by cosinor analysis. Bias and limits of agreement of means and median HR with mesor HR were determined by correlation and Bland-Altman analysis., Results: Circadian variation in HR was identified not only in 18/22 healthy dogs and 14/21 AF dogs but only on ambulatory electrocardiogram recordings. Four-hourly spot-checks provided the most accurate estimate of mesor HR in healthy dogs (bias of the median over 30 s 7.70, limits of agreement 7.48), whereas, in dogs with AF, four, six and eight-hourly spot-checks provided reliable estimates of mesor HR (bias within -1.29 and -29.5)., Conclusions: Four, six and eight-hourly HR spot-checks can estimate 24-hourly HR in dogs with AF. There was CV in HR in most healthy pet dogs and dogs with AF. Spot-check protocols cannot identify CV in HR., Competing Interests: Conflict of Interest Statement The authors do not have any conflicts of interest to disclose., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)