Background: Salbutamol bronchodilator response (BDR) test and fractional exhaled nitric oxide (FeNO) have been recommended for the diagnosis of asthma in children, but FeNO levels is affected by many factors.Nonetheless, data of the effect on the FeNO values throughout the bronchodilator test and the differences in FeNO values between BDR positive (BDR+) and negative (BDR−) children with asthma are still limited. We aimed to evaluate the effect of the BDR test on FeNO and the differences in FeNO levels between BDR+ and BDR− children with asthma.Methods: This was a prospective, observational study performed over a 5-month period (December 2018 to April 2019) and involved 57 children with asthma. The FeNO levels at pre-spirometry, post-spirometry, and post-salbutamol BDR testing were estimated. Finally, the children were divided into two groups i.e., BDR+ and BDR−, and differences in the FeNO levels were compared between the two groups.Results: The spirometry results were normal in 2 patients (3.5%). There were 53 (93%) patients with obstructive lung disease, including 40 (70.2%), 11 (19.3%), and 2 (3.5%) patients with mild, moderate, and severe obstruction, respectively. The remaining two patients had mixed lesions (3.5%), none of which were restrictive. The baseline median FeNO levels were significantly higher in the BDR+ group than in the BDR− group [33.00 (23.78, 46.73) vs. 23.00 (9.80, 37.80), (P=0.048)]. Following spirometry, there was a statistically significant decrease in median FeNO levels from baseline to post-spirometry (P=0.002). However, there was no significant difference between the median FeNO levels at baseline and following the BDR test (P=0.976).The impact of spirometry on FeNO was not statistically different in BDR+ versus BDR− children (Z=−0.186, P=0.853); however, the impact of bronchodilators on FeNO exhibited a statistically significant difference between the two groups (Z=3.160, P=0.002).Conclusions: This study revealed dynamic changes in the FeNO levels during the BDR test. The use of a bronchodilator results in a statistically significant difference in FeNO levels between BDR+ and BDR− children with asthma. Moreover, spirometry leads to a marked decrease in the FeNO levels. Our results will allow clinicians to better interpret FeNO, BDR and pulmonary function outcomes and better develop clinical protocols.