The pH measurement of exhaled breath condensate (EBC) may provide a non-invasive method of assessing the lower airways of horses but the methodology used may influence findings. The aim of this study was to investigate the effect of two sampling devices and three methods of condensation surface cooling (ethanol slush, '100 °C; dry ice, '75 °C; water ice, 0 °C) on EBC pH. Each method was tested 30 times using six healthy ponies. Sample pH was determined before and after de-aeration with argon for 10 min.Sampler design was found to significantly affect pH. Samples collected as a liquid had a significantly higher pH than samples frozen during collection (P < 0.05). De-aeration resulted in significantly higher pH (P < 0.05) with less variation. This study has shown that device design and condensation surface temperature will influence EBC pH, which will prevent a direct comparison of results when different methodologies are used.