The vasovagal tonus index (VVTI), a time-domain indicator of heart rate variability, has been suggested as a diagnostic and prognostic tool in dogs with cardiac disease and heart failure. Brachycephalic breeds tend to have a higher VVTI than non-brachycephalic breeds.11 The objective of this study was to obtain standard VVTI reference values from a representative cohort of healthy French Bulldogs based on a single ECG recording. A second aim of this study was to evaluate the practicability of a VVTI screening in the clinical routine by evaluating repeatability of the VVTI across five successive measurements. In order to determine baseline factors potentially influencing the VVTI, the impact of stress, activity and character as well as sex and neutering status were evaluated. Out of 73 French Bulldogs screened, 60 dogs were considered healthy based on their medical history, physical examination, blood pressure and ECG, and no evidence of congenital or acquired heart disease on echocardiographic examination. The VVTI was calculated based on the variance of 20 R-R intervals and the corresponding HR extrapolated to one minute from the same 20 R-R intervals. In addition, a maximum of five consecutive VVTI values were calculated based on 100 successive R-R intervals recorded from each dog. VVTI values ranged from 5.66 to 11.3 with a mean (standard deviation [SD]) of 8.82 (1.43); the corresponding HR ranged from 78 to 173 bpm with a mean (SD) of 120 (23) bpm. Importantly, VVTI and HR values were negatively correlated (R = 0.689; adjusted R(2) = 0.466), which must be considered for clinical interpretation of the VVTI. The repeatability across five successive sets of 20 heartbeats was shown (mean intra-individual variability of 6.1%). Stress significantly influenced the VVTI and HR (p < 0.001). The VVTI range established in this study may be used as reference to assess the HRV of presumably healthy brachycephalic dogs at routine health checks. Deviations from the reference may permit the clinician to adapt the schedule and focus of subsequent follow-up investigations.