Multiple breath washout (MBW) for measuring the lung clearance index (LCI) has been proposed as a non-invasive tool for detecting early cystic fibrosis (CF) lung disease. The LCI is highly repeatable and reproducible in healthy subjects. In patients with CF, within-test variability is low. However, application of physiotherapy (PT) immediately preceding MBW may affect LCI variability in CF patients and thus interpretation of repeat measurements and treatment effects. Therefore, the aim of the present study was to prospectively assess the short-term effect of PT on LCI in CF patients in order to address the question whether or not standardized timing of PT and MBW has to be considered when introducing MBW into clinical CF management. Twenty-seven out of 32 patients (5.7-15.9 years) with CF successfully performed two technically acceptable MBW tests with the EasyOne Pro, MBW Module (ndd, Switzerland) at intervals of 1(1/2) hr. Sixteen out of 27 received 30 min PT in between, whereas 11/27 did not.Repeatability expressed as intraindividual coefficient of variation (CV) was 6.1% pre-PT and 6.5% post-PT. Mean difference (95% CI) of LCI between the two tests was -0.20 (-0.51; 0.11). Reproducibility (SD) was 4.6% (3.1). Repeatability was 4.2% and 7.1% without intervention. Mean difference (95% CI) of LCI between 1st and 2nd test was 0.07 (-0.22; 0.35). Reproducibility (SD) was 2.6% (2.1).In conclusion, PT does not have a consistent effect on the LCI. Repeatability was slightly poorer than published for healthy subjects possibly reflecting variable mucus plugging, and, thus, variable trapped air in patients with CF. Reproducibility was good and independent on intervention. From our data, we conclude that timing of PT in relation to MBW can be ignored when designing study protocols or when interpreting longitudinal data and treatment effects.