Introduction: Diaphragm ultrasound is increasingly used in adults, and more recently in pediatric practice. However, normal diaphragm parameters in healthy infants are unknown. This was a prospective observational pilot study aiming to define the normal diaphragm ultrasound characteristics in healthy infants during the first 6 months of life. Methods: We recruited healthy neonates at 7 to 15 days of life, who were followed until the sixth month of life, undergoing five assessments in different time points. The measurements included diaphragm thickness at end expiration (TEE) and at end inspiration (TEI). The thickening fraction (TF) was calculated as (TEI-TEE)/TEE and expressed as a percentage, and as (TEI-TEE)/TEI. Results: A total of 37 toddlers, 16 of which were females (43.2%), were enrolled. Thirty-four children (91.9%) were of Caucasian ethnicity and the median gestational age was 38.4 (35.7–40) weeks. Normal TEE, TEI, and TF have been provided for each time point. Conclusion: We provided new insight regarding data about thickness and thickening function in healthy children to be used for future physiologic and pathologic pediatric studies.