BACKGROUND: A 20% reduction in the FEV 1 is routinely used as an end point for methacholine challenge testing (MCT). Measurement of FEV 1 is effort dependent, and some patients are not able to perform acceptable and repeatable forced expiration maneuvers. The goal of the present study was to investigate the diagnostic value of airway resistance measurement by forced oscillation technique (FOT), body plethysmography, and interrupter technique compared with the traditionally accepted standard FEV 1 measurement in evaluating the responsiveness to methacholine during MCT. METHODS: We included in the study adult subjects referred for MCT because of asthma-like symptoms and with normal baseline spirometry. We modified routine MCT protocol by adding the assessment of airway resistance to the measurement of FEV 1 at each step of MCT. RESULTS: We observed, in the subjects with airway hyper-responsiveness versus those with normal airway responsiveness, a significantly greater percentage change in median (interquartile range) FOT resistance at 10 Hz (25.9% [13.7%–35.4%] vs 16% [15.7%–27.2%]), plethysmographic resistance (70.2% [39.5%–116.3%] vs 37.1% [23.9%–81.9%]), and mean ± SD conductance (−41.3 ± 15.4% vs −29.6 ± 15.9%); and a significantly greater change in mean ± SD FOT reactance at 10 Hz (–0.41 ± 0.48 cm H 2 O/L/s vs –0.09 ± 0.32 cm H 2 O/L/s) and at 15 Hz (–0.29 ± 0.2 cm H 2 O/L/s vs –0.1 ± 0.19 cm H 2 O/L/s). We also recorded significant differences in airway resistance parameters (FOT resistance at 10 Hz, FOT reactance at 15 Hz, plethysmographic airway resistance, and conductance indices as well as interrupter resistance) in FEV 1 non-responders at the onset of respiratory symptoms during MCT compared with baseline. CONCLUSIONS: Measurements of airway resistance could possibly be used as an alternative method to spirometry in airway challenge. Significant changes in airway mechanics during MCT are detectable by airway resistance measurement in FEV 1 non-responders with methacholine-induced asthma-like symptoms. (ClinicalTrials.gov registration NCT02343419.)