Vagus nerve stimulation (VNS) is an established treatment for epilepsy and depression in human patients, but in both humans and dogs, optimal stimulation parameters remain unknown. Delivering afferent bursts of stimulation may be promising as a means of increasing efficacy, but evaluation of potential effects on the heart due to unavoidable efferent stimulation is required. The present study investigated heart rate variability (HRV) in healthy Beagle dogs treated with 1 h of sham, standard or microburst left-sided VNS in a crossover design. No significant differences were found between the stimulation paradigms for any of the cardiac parameters. Short-term left-sided VNS, including a novel bursting pattern (microburst VNS), had no statistically significant effect on HRV in ambulatory healthy dogs. Studies in a larger number of animals with long-term VNS are recommended.