Background: Respiratory problems are common in horses, and are often diagnosed as a cause of poor athletic performance. Reliable, accurate and sensitive spirometric tests of airway function in resting horses would assist with the diagnosis of limitations to breathing and facilitate investigations of the effects of various treatments on breathing capacity. The evaluation of respiratory function in horses is challenging and suitable procedures are not widely available to equine practitioners. The determination of relative flow or flow-time measures is used in paediatric patients where compliance may limit conventional pulmonary function techniques. The aim of the current study was to characterise absolute and relative indices of respiratory function in healthy horses during eupnoea (tidal breathing) and carbon dioxide (CO2)-induced hyperpnoea (rebreathing) using a modified mask pneumotrachographic technique well suited to equine practice, and to evaluate the reliability of this technique over three consecutive days. Coefficients of variation, intra-class correlations, mean differences and 95% confidence intervals across all days of testing were established for each parameter. Results: The technique provided absolute measures of respiratory function (respiratory rate, tidal volume, peak inspiratory and expiratory flows, time to peak flow) consistent with previous studies and there was no significant effect of day on any measure of respiratory function. Variability of measurements was decreased during hyperpnea caused by rebreathing CO2, but a number of relative flow-time variables demonstrated good agreement during eupnoeic respiration. Conclusions: The technique was well tolerated by horses and study findings suggest the technique is suitable for evaluation of respiratory function in horses. The use of relative flow-time variables provided reproducible (consistent) results, suggesting the technique may be of use for repeated measures studies in horses during tidal breathing or rebreathing.