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Background Neurally adjusted ventilatory assist (NAVA) delivers airway pressure (Paw) in proportion to neural inspiratory drive as reflected by electrical activity of the diaphragm (EAdi). Changing positive end-expiratory pressure (PEEP) impacts respiratory muscle load and function and, hence, EAdi. We aimed to evaluate how PEEP affects the breathing pattern and neuroventilatory efficiency during NAVA. Methods In 20 adult patients, adequate assist (NAVAal) was first identified based on Paw and tidal volume (V t ) responses to systematic increases in NAVA level while using preset PEEP (PEEPbl). Thereafter, using NAVAal, PEEP was increased to 20 cm water (H 2 O) (PEEPhigh) and then lowered stepwise to 1 cm H 2 O (PEEP1). EAdi, Paw, and V t were recorded. Results Median NAVAal was 2.7 (interquartile range, 2.3–3.5) cm H 2 O/μV and was similar to NAVAal identified post hoc by 17 independent physicians (2.5 [2.0–3.4] cm H 2 O/μV; P = NS). Reducing PEEPhigh to PEEP1 increased inspiratory EAdi by 34% (2–67; P = .046) and was associated with an increase in mean Paw above PEEP from 8.5 (6.7–11.4) cm H 2 O to 12.2 (8.8–16.7) cm H 2 O ( P = .008), whereas V t and respiratory rate remained unchanged. The response pattern in V t /EAdi, indicating changes in neuroventilatory efficiency, differed among patients. Tidal breathing occurred at the lowest EAdi cost in seven patients with PEEP1 or half PEEPbl, in six patients with PEEPbl, and in seven patients with PEEPhigh. Conclusions During NAVAal, increasing PEEP reduces respiratory drive. Patients adapt their neuroventilatory efficiency such that the individual ventilatory pattern is preserved over a wide range of PEEP levels. Monitoring V t /EAdi during PEEP changes allows identification of a PEEP level at which tidal breathing occurs at minimal EAdi cost. Trial registration clinicaltrials.gov ; Identifier: NCT00529347