Background: Direct inhalation challenges (e.g. methacholine) are stated to be more sensitive and less specific for a diagnosis of asthma than are indirect challenges (e.g. exercise, non-isotonic aerosols, mannitol, etc.). However, data surrounding comparative sensitivity and specificity for methacholine compared to mannitol challenges are conflicting. When methacholine is inhaled by deep total lung capacity (TLC) inhalations, deep inhalation inhibition of bronchoconstriction leads to a marked loss of diagnostic sensitivity when compared to tidal breathing (TB) inhalation methods. We hypothesized that deep inhalation methacholine methods with resulting bronchoprotection may be the explanation for conflicting sensitivity/specificity data.Methods: We reviewed 27 studies in which methacholine and mannitol challenges were performed in largely the same individuals. Methacholine was inhaled by dosimeter TLC methods in 13 studies and by tidal breathing in 14 studies. We compared the rates of positive methacholine (stratified by inhalation method) and mannitol challenges in both asthmatics and non-asthmatics.Results: When methacholine was inhaled by TLC inhalations the prevalence of positive tests in asthmatics, 60.2% (548/910), was similar to mannitol, 58.9% (537/912). By contrast, when methacholine was inhaled by tidal breathing the prevalence of positive tests in asthmatics 83.1% (343/413) was more than double that of mannitol, 41.5% (146/351). In non-asthmatics, the two methacholine methods resulted in positive tests in 18.8% (142/756) and 16.2% (27/166) by TLC and TB inhalations respectively. This compares to an overall 8.3% (n = 76) positive rate for mannitol in 913 non-asthmatics.Conclusion: These data support the hypothesis that the conflicting data comparing methacholine and mannitol sensitivity and specificity are due to the method of methacholine inhalation. Tidal breathing methacholine methods have a substantially greater sensitivity for a diagnosis of asthma than either TLC dosimeter methacholine challenge methods or mannitol challenge. Methacholine challenges should be performed by tidal breathing as per recent guideline recommendations. Methacholine (more sensitive) and mannitol (more specific) will thus have complementary diagnostic features.