Diaphragmatic thickness (Tdi) and diaphragm thickening fraction (dTF) are widely used parameters in ultrasound studies of the diaphragm in mechanically ventilated children, but normal values for healthy children are scarce. We determined reference values of Tdi and dTF using ultrasound in healthy children aged 0-8 years old and assessed their reproducibility. In a prospective, observational cohort, Tdi and dTF were measured on ultrasound images across four age groups comprising at least 30 children per group: group 1 (0-6 months), group 2 (7 months-1 year), group 3 (2-4 years) and group 4 (5-8 years). Ultrasound images of 137 healthy children were included. Mean Tdi at inspiration was 2.07 (SD 0.40), 2.09 (SD 0.40), 1.69 (SD 0.30) and 1.72 (SD 0.30) mm for groups 1, 2, 3 and 4, respectively. Mean Tdi at expiration was 1.64 (SD 0.30), 1.67 (SD 0.30), 1.38 (SD 0.20) and 1.42 (SD 0.20) mm for groups 1, 2, 3 and 4, respectively. Mean Tdi at inspiration and mean Tdi at expiration for groups 1 and 2 were significantly greater than those for groups 3 and 4 (both p < 0.001). Mean dTF was 25.4% (SD 10.4), 25.2% (SD 8.3), 22.8% (SD 10.9) and 21.3% (SD 7.1) for group 1, 2, 3 and 4, respectively. The intraclass correlation coefficients (ICC) representing the level of inter-rater reliability between two examiners performing the ultrasounds was 0.996 (95% CI 0.982-0.999). ICC of the inter-rater reliability between the raters in 11 paired assessments was 0.989 (95% CI 0.973-0.995).   Conclusion: Ultrasound measurements of Tdi and dTF were highly reproducible in healthy children aged 0-8 years.    Trial registration: ClinicalTrials.gov identifier (NCT number): NCT04589910. What is Known: • Diaphragmatic thickness and diaphragm thickening fraction are widely used parameters in ultrasound studies of the diaphragm in mechanically ventilated children, but normal values for healthy children to compare these with are scarce. What is New: • We determined normal values of diaphragmatic thickness and diaphragm thickening fraction using ultrasound in 137 healthy children aged 0-8 years old. The diaphragmatic thickness of infants up to 1 year old was significantly greater than that of children from 2 to 8 years old. Diaphragmatic thickness decreased with an increase in body surface area. These normal values in healthy children can be used to assess changes in respiratory muscle thickness in mechanically ventilated children.