Guidelines for the measurement of fractional exhaled nitric oxide (FENO) recommend refraining from lung function tests (LFT) and certain foods and beverages before performing FENO measurements, as they may lead to transiently altered FENO levels. Little is known of such factors in infants. The aim of the present study was to evaluate whether forced expiratory maneuvers, sedation, nasal contamination, and breastfeeding affect FENO values in infants. FENO was measured off-line during tidal breathing by means of a facemask covering nose and mouth. FENO measurements were performed in 45 sedated infants (mean age 12.1 months) who underwent LFT because of airway diseases and in 83 unsedated healthy infants (mean age 4.3 months). In infants with airway diseases, no difference was found in FENO values before and 5 min after LFT (n = 19 infants, p = 0.7) and FENO values before sedation did not differ from FENO values during sedation (n = 10 infants, p = 0.2).Oral FENO values were significantly lower than mixed (nasal + oral) FENO (n = 42 infants, p < 0.001). FENO values before and 5 min after breastfeeding were not different (n = 11 healthy infants, p = 0.57). The short-term reproducibility in healthy infants (n = 54) was satisfactory (intraclass correlation coefficient = 0.94). We conclude that, in infants with airway diseases, LFT prior to FENO measurement did not influence FENO values and FENO values did not change after sedation. Oral FENO values were significantly lower than mixed (oral + nasal) FENO, and breastfeeding did not influence FENO. Short-term reproducibility in awake healthy infants was good.