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Objectives: The objective of the study is to determine the impact of changing reference standards (RS), namely spirometry vs. whole-body plethysmography (WBP), on estimation of the diagnostic accuracy of fractional exhaled nitric oxide (FeNO) and clinical signs and symptoms (CSS) as index tests regarding asthma diagnosis., Study Design and Setting: This was a diagnostic study conducted in 393 patients attending a private practice of pneumologists with complaints suspicious of asthma. First, the index tests were compared with the diagnostic results of spirometry in terms of forced expiratory volume in the first second (FEV 1 ) responsiveness. Second, the index tests were compared with the results of WBP in terms of specific airway resistance and FEV 1 responsiveness. Areas under the curve (AUC) were compared with a generalized estimating equation approach based on binary logistic regression., Results: FeNO values and CSS 'wheezing' and 'allergic rhinitis' showed higher specificities (P < 0.001) and sensitivities (not significant) when evaluated with WBP; also, Youden indices increased in these CSS (P < 0.05). AUC of FeNO in combination with 'wheezing' and 'allergic rhinitis' when WBP was used as RS (AUC = 0.724; 95% confidence interval 0.672 to 0.776) was higher compared with spirometry as RS (AUC = 0.654; 95% confidence interval 0.585 to 0.722) (P < 0.001)., Conclusion: In case of asthma, superior RS led to more favorable assessment of index tests. FeNO measurement might have been underestimated in some previous studies., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)