Exhaled nitric oxide (ENO) is used increasingly as a surrogate marker of airway inflammation in research protocols that may incorporate standard efficacy measures, such as spirometry before and after bronchodilator, which could affect ENO measurements. In seven healthy volunteers and 11 mild asthmatic subjects, we measured ENO before and serially for 1 h after spirometry. On two additional days in the subjects with asthma, we reexamined the effect of spirometry as before, followed by the serial measurement of ENO for 1 h after two puffs of salbutamol (100 microgram/puff) by metered-dose inhaler or matching placebo. As early as 1 min after spirometry, ENO fell by 13% and 10% in the normal and asthmatic subjects, respectively. In both groups, ENO returned to baseline over 1 h. In the asthmatic subjects, salbutamol caused a significant mean increase of the order of 10 parts per billion in ENO (p < 0.001) for 1 h as compared with placebo inhaler. We conclude that spirometry and beta2-agonist may perturb ENO values and recommend that studies control for these factors.