Elevated levels of exhaled nitric oxide at a flow of 50 mL·s⁻¹(F_ENO₅₀) are an important indicator of Th2 airway inflammation and may aid clinicians in the diagnosis and monitoring of asthma. This study aimed to derive Global Lung Function Initiative reference equations and the upper limit of normal for F_ENO₅₀.Available individual F_ENO₅₀data were collated and harmonised using consensus-derived variables and definitions. Data collected from individuals who met the harmonised definition of “healthy” were analysed using generalised additive models of location shape and scale (GAMLSS) technique.Data were retrospectively collated from 34 782 individuals from 34 sites in 15 countries, of whom 8022 met the definition of healthy (19 sites, 11 countries). Overall, height, age and sex only explained 12% of the between-subject variability of F_ENO₅₀(R²=0.12). The addition of NO device was a predictor of F_ENO₅₀and between-subject variability, such that the healthy range of values and the upper limit of normal varied depending on which device was used. The range of F_ENO₅₀values observed in healthy individuals was also very wide, and the heterogeneity was partially explained by the device used. The heterogeneity between sites remained within a sub-set of data where F_ENO₅₀was measured using the same device and a stricter definition of health (n=1027).Available F_ENO₅₀data collected from different sites using different protocols and devices was too variable to develop a single all-age reference equation. Further standardisation of NO devices and measurement is required before population reference values might be derived.