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Objective—To assess the variability in vertebral heart score (VHS) measurement induced by cardiac and respiratory cycles in dogs. Design—Prospective observational study. Animals—14 healthy Beagles. Procedures—Dogs underwent fluoroscopic examination by 4 observers, and VHS was measured at end-tidal inspiration and end-tidal expiration during end systole and end diastole in left and right lateral recumbency. Mean VHS was compared within and among cardiac and respiratory phases and recumbency type, and correlation between VHS and heart rate was investigated. Interobserver variability was assessed. Results—Mean VHS for each combination of respiratory and cardiac cycle was larger on images obtained in right lateral versus left lateral recumbency. The greatest differences were observed between VHS measured in the diastolic inspiratory phase (mean ± SD, 10.59 ± 0.49 vertebral units [VU] and 10.35 ± 0.50 VU for right and left lateral recumbency, respectively) and the systolic expiratory phase (10.11 ± 0.37 VU and 9.92 ± 0.50 VU for right and left lateral recumbency, respectively). The combination of respiratory and cardiac cycles induced a maximal difference in VHS of up to 0.97 VU and 1.11 VU in the inspiratory and expiratory phases, respectively. Heart rate was not correlated with the difference between VHS in systolic and diastolic phases. Conclusions and Clinical Relevance—Clinicians should be aware of the potential influence of these factors when assessing VHS in dogs; in addition to allowing optimal pulmonary assessment, consistently taking radiographs at end-inspiratory tidal volume may help to limit VHS variability attributable to the respiratory cycle. Further research is needed to assess the effects of cardiac and respiratory phases on VHS in dogs with cardiac or respiratory disease.