Purpose: To investigate the development of diaphragmatic dysfunction in ventilated extremely preterm infants (EPI) using diaphragm ultrasound (DU). Methods: EPI of less than 28 weeks' gestational age who required mechanical ventilation within six hours of birth were included in this prospective, observational study. The DU was performed once a day until four days of life. End-inspiratory and end-expiratory thicknesses of the diaphragm were measured and the diaphragm thickening fraction was calculated.Results: A total of 20 EPI were enrolled. After intubation, there was a progressive reduction in endinspiratory thicknesses of the diaphragm from baseline to day 1 (P < 0.001), but not from day 1 to day 2 (P = 0.078), day 2 to day 3 (P = 1.0), or day 3 to day 4 (P = 1.0). There was also a signi cant reduction in the diaphragm thickening fraction from baseline to day 1 (P < 0.001), but not from day 1 to day 2 (P = 1.0), day 2 to day 3 (P = 1.0), or day 3 to day 4 (P = 1.0).Conclusions: This study provides the rst evidence of diaphragmatic dysfunction in ventilated EPI. We demonstrated a rapid progression of ventilator-induced diaphragmatic dysfunction, with a signi cant reduction in diaphragm thickness and thickening fraction within 24 hours of ventilation.