Abstract Aim Exhaled Nitric oxide (eNO) is an inflammatory marker. In 2002 Hall et al. [J Appl Physiol. 92:59?66] established an infant eNO measurement method, fulfilling four criteria of feasibility: simple, non?invasive, without impact on the natural breathing pattern, and accounting for flow by NO output (V'NO). Although tidal breathing is accepted as an eNO measurement method in uncooperative patients, it is seldom used outside research labs. The variability and lack of validated methods have restrained from exploring the area in preterm and term neonates the last years. This study aimed to validate clinically feasible longitudinal online tidal eNO and V'NO in a real?life birth cohort of un?sedated, hospitalized preterm, and term neonates. Method We included 149 newborns, GA 28?42 weeks. Each scheduled for six repeated, non?invasive, on?line eNO measurements with Ecomedics CLD 88sp and NO?free air. We used three 60?second?eNO measurements. The method was adapted to fit preterm and term neonates with unstable respiration, without excluding sighs and surrounding breaths. Result Protocol measurements with a maximum mutual difference of 1?ppb succeeded in 85?99%, increasing with postnatal age. We performed mixed model analyses in three hierarchical measurement levels. Despite the irregular breathing of newborns, the predictions of individual eNO levels in the average infant was a 0.05?SD. Exhaled NO was flow?dependent (P?=?0.028); V'NO but not eNO was associated with preterm birth (P?24?h CPAP treatment (P?=?0.0316). Conclusion We validated clinically, non?invasive, online eNO measurements in neonates. The method was well tolerated and exhibited low subject?specific?prediction?variance and high success rates.