Abstract Background The effects of changes in cooling temperature on biomarker levels in exhaled breath condensate have been little investigated. The aim of the study was to test the effect of condensation temperature on the parameters of exhaled breath condensate and the levels of selected biomarkers. Methods Exhaled breath condensate was collected from 24 healthy subjects at temperatures of -10, -5, 0 and +5 C degrees. Selected parameters (condensed volume and conductivity) and biomarkers (hydrogen peroxide, malondialdehyde) were measured. Results There was a progressive increase in hydrogen peroxide and malondialdehyde concentrations, and condensate conductivity as the cooling temperature increased; total condensate volume increased as the cooling temperature decreased. Conclusion The cooling temperature of exhaled breath condensate collection influenced selected biomarkers and potential normalizing factors (particularly conductivity) in different ways ex vivo. The temperature of exhaled breath condensate collection should be controlled and reported.