Background: Ventilation with lower positive end–expiratory pressure (PEEP) may cause loss of lung aeration in critically ill invasively ventilated patients. This study investigated whether a systematic lung ultrasound (LUS) scoring system can detect such changes in lung aeration in a study comparing lower versus higher PEEP in invasively ventilated patients without acute respiratory distress syndrome (ARDS). Methods: Single center substudy of a national, multicenter, randomized clinical trial comparing lower versus higher PEEP ventilation strategy. Fifty–seven patients underwent a systematic 12–region LUS examination within 12 h and between 24 to 48 h after start of invasive ventilation, according to randomization. The primary endpoint was a change in the global LUS aeration score, where a higher value indicates a greater impairment in lung aeration. Results: Thirty–three and twenty–four patients received ventilation with lower PEEP (median PEEP 1 (0–5) cm H2O) or higher PEEP (median PEEP 8 (8–8) cm H2O), respectively. Median global LUS aeration scores within 12 h and between 24 and 48 h were 8 (4 to 14) and 9 (4 to 12) (difference 1 (–2 to 3)) in the lower PEEP group, and 7 (2–11) and 6 (1–12) (difference 0 (–2 to 3)) in the higher PEEP group. Neither differences in changes over time nor differences in absolute scores reached statistical significance. Conclusions: In this substudy of a randomized clinical trial comparing lower PEEP versus higher PEEP in patients without ARDS, LUS was unable to detect changes in lung aeration.