Abstract Background Exercise testing to aid diagnosis of exercise-induced bronchoconstriction (EIB) is commonly performed. Reproducibility of the airway response to a standardized exercise protocol has not been reported in subjects being evaluated with mild symptoms suggestive of asthma but without a definite diagnosis. This study examined reproducibility of % fall in FEV1 and area under the FEV1 time curve for 30 minutes in response to two exercise tests performed with the same intensity and duration of exercise, and inspired air conditions. Methods Subjects with mild symptoms of asthma exercised twice within approximately 4 days by running for 8 minutes on a motorized treadmill breathing dry air at an intensity to induce a heart rate between 80-90% predicted maximum; reproducibility of the airway response was expressed as the 95% probability interval. Results Of 373 subjects challenged twice 161 were positive (≥10% fall FEV1 on at least one challenge). The EIB was mild and 77% of subjects had <15% fall on both challenges. Agreement between results was 76.1% with 56.8% (212) negative (< 10% fall FEV1) and 19.3% (72) positive on both challenges. The remaining 23.9% of subjects had only one positive test. The 95% probability interval for reproducibility of the % fall in FEV1 and AUC0-30 min was ± 9.7% and ± 251% for all 278 adults and ± 13.4% and ± 279% for all 95 children. The 95% probability interval for reproducibility of % fall in FEV1 and AUC0-30 min for the 72 subjects with two tests ≥10% fall FEV1 was ± 14.6% and ± 373% and for the 34 subjects with two tests ≥15% fall FEV1 it was ± 12.2% and ± 411%. Heart rate and estimated ventilation achieved were not significantly different either on the two test days or when one test result was positive and one was negative. Conclusions Under standardized, well controlled conditions for exercise challenge, the majority of subjects with mild symptoms of asthma demonstrated agreement in test results. Performing two tests may need to be considered when using exercise to exclude or diagnose EIB, when prescribing prophylactic treatment to prevent EIB and when designing protocols for clinical trials.