Heart rate variability (HRV) is a physiologic phenomenon that occurs due to changing autonomic tone resulting in variable RR intervals. A reduction in HRV is used as an index of pain in neonatal human patients. Objective measures of pain would be valuable in the evaluation of canine patients and assessment of response to pain management strategies. We hypothesized that dogs with diseases associated with discomfort (osteoarthritis and bone neoplasia) would have reduced HRV compared with normal, healthy dogs. The aim of the study was to calculate the sample size necessary to investigate this hypothesis. Seventeen dogs from the Ryan Veterinary Hospital of the University of Pennsylvania patient population or owned by Ryan Veterinary Hospital of the University of Pennsylvania staff were enrolled in this single-blind, prospective pilot study. A 30 min electrocardiogram (ECG) was obtained from each dog using an ambulatory electrocardiographic monitor. All ECGs were obtained between 10 a.m. and 2 p.m. ECGs were analyzed and time-domain HRV indices computed. Sample size calculations suggest that 207 dogs would be necessary to ascertain if HRV is reduced in dogs experiencing discomfort or pain (50 in the arthritis group, 79 in the bone cancer group, and 78 in the control group).