Background: Neurally adjusted ventilatory assist (NAVA) is an emerging mode of respiratory support that uses the electrical activity of the diaphragm (Edi) to provide synchronised inspiratory pressure support, proportional to an infant’s changing inspiratory effort. Data on Edi reference values for neonates are limited. The objective of this study was to establish reference Edi values for preterm and term neonates. Methods: This was a prospective observational study of newborn infants breathing spontaneously in room air. The Edi signal was monitored by a specialised intragastric feeding tube with embedded electrodes positioned at the level of the diaphragm. Edi minimums and peaks were recorded continuously for four hours. Results: 24 newborn infants (16 preterm [<37 weeks’ gestation]; 8 term) were studied. All infants were breathing comfortably in room air at the time of study. Edi data were successfully captured in all infants. The mean (±SD) Edi minimum was 3.02 (±0.94) µV and the mean Edi peak was 10.13 (±3.50) µV. In preterm infants the mean (±SD) Edi minimum was 3.05 (±0.91) µV and the mean Edi peak was 9.36 (±2.13) µV. In term infants the mean (±SD) Edi minimum was 2.97 (±1.05) µV and the mean Edi peak was 11.66 (±5.14) µV. Conclusion: Reference Edi values were established for both preterm and term neonates. These values can be used as a guide when using diaphragm-triggered modes on respiratory support in newborn infants.