Background: Acute increases in muscle sonographic echodensity reflect muscle injury. Diaphragm echodensity has not been measured in mechanically ventilated patients. We undertook to develop a technique to characterize changes in diaphragm echodensity during mechanical ventilation and to assess whether these changes are correlated with prolonged mechanical ventilation. Methods: Diaphragm ultrasound images were prospectively collected in mechanically ventilated patients and in 10 young healthy subjects. Echodensity was quantified based on the right-skewed distribution of grayscale values (50th percentile, ED50; 85th percentile, ED85). Intra- and inter-analyzer measurement reproducibility was determined. Outcomes recorded included duration of ventilation and ICU complications (including reintubation, tracheostomy, prolonged ventilation, or death). Results: Echodensity measurements were obtained serially in 34 patients comprising a total of 104 images. Baseline (admission) diaphragm ED85 was increased in mechanically ventilated patients compared to younger healthy subjects (median 56, interquartile range (IQR) 42-84, vs. 39, IQR 36-52, p = 0.04). Patients with an initial increase in median echodensity over time (≥ + 10 in ED50 from baseline) had fewer ventilator-free days to day 60 (n = 13, median 46, IQR 0-52) compared to patients without this increase (n = 21, median 53 days, IQR 49-56, unadjusted p = 0.03). Both decreases and increases in diaphragm thickness during mechanical ventilation were associated with increases in ED50 over time (adjusted p = 0.03, conditional R2 = 0.80) and the association between increase in ED50 and outcomes persisted after adjusting for changes in diaphragm thickness. Conclusions: Many patients exhibit increased diaphragm echodensity at the outset of mechanical ventilation. Increases in diaphragm echodensity during the early course of mechanical ventilation are associated with prolonged mechanical ventilation. Both decreases and increases in diaphragm thickness during mechanical ventilation are associated with increased echodensity.