Airway epithelial injury is regarded as a key contributing factor to the pathogenesis of exercise-induced bronchoconstriction (EIB) in athletes. The concentration of the pneumoprotein club cell (Clara cell) CC16 in urine has been found to be a non-invasive marker for hyperpnoea-induced airway epithelial perturbation. Exercise-hyperpnoea induces mechanical, thermal and osmotic stress to the airways. We investigated whether osmotic stress alone causes airway epithelial perturbation in athletes with suspected EIB. Twenty-four recreational summer sports athletes who reported respiratory symptoms on exertion performed a standard eucapnic voluntary hyperpnoea test with dry air and a mannitol test (osmotic challenge) on separate days. Median urinary CC16 increased from 120 to 310 ρg μmol creatinine⁻¹ after dry air hyperpnoea (P = 0.002) and from 90 to 191 ρg μmol creatinine⁻¹ after mannitol (P = 0.021). There was no difference in urinary CC16 concentration between athletes who did or did not bronchoconstrict after dry air hyperpnoea or mannitol. We conclude that, in recreational summer sports athletes with respiratory symptoms, osmotic stress per se to the airway epithelium induces a rise in urinary excretion of CC16. This suggests that hyperosmolarity of the airway surface lining perturbs the airway epithelium in symptomatic athletes.