Objective To determine the impact of changing reference standards (RS), namely spirometry versus whole body plethysmography (WBP), on estimation of the diagnostic accuracy of Fractioned exhaled Nitric Oxide (FeNO) and clinical signs and symptoms (CSS) as index tests regarding asthma diagnosis. Study design and setting Diagnostic study in 393 patients attending a private practice of pneumologists with complaints suspicious of asthma. Firstly, the index tests were compared to the diagnostic results of spirometry in terms of FEV1 responsiveness. Secondly, the index tests were compared to the results of WBP in terms of specific airway resistance and FEV1 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%CI 0.672 to 0.776) was higher compared to spirometry as RS (AUC=0.654; 95%CI 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.