Abstract Background Respiratory effort should be closely monitored in mechanically ventilated ICU patients to avoid both overassistance and underassistance. Surface electromyography of the diaphragm (sEMGdi) offers a continuous and non-invasive modality to assess respiratory effort based on neuromuscular coupling (NMCdi). The sEMGdi derived electrical activity of the diaphragm (sEAdi) is prone to distortion by crosstalk from other muscles including the heart, hindering its widespread use in clinical practice. We developed an advanced analysis as well as quality criteria for sEAdi waveforms and investigated the effects of clinically relevant levels of PEEP on non-invasive NMCdi. Methods NMCdi was derived by dividing end-expiratory occlusion pressure (Pocc) by sEAdi, based on three consecutive Pocc manoeuvres at four incremental (+ 2 cmH2O/step) PEEP levels in stable ICU patients on pressure support ventilation. Pocc and sEAdi quality was assessed by applying a novel, automated advanced signal analysis, based on tolerant and strict cut-off criteria, and excluding inadequate waveforms. The coefficient of variations (CoV) of NMCdi after basic manual and automated advanced quality assessment were evaluated, as well as the effect of an incremental PEEP trial on NMCdi. Results 593 manoeuvres were obtained from 42 PEEP trials in 17 ICU patients. Waveform exclusion was primarily based on low sEAdi signal-to-noise ratio (N tolerant = 155, 37%, N strict = 241, 51% waveforms excluded), irregular or abrupt cessation of Pocc (N tolerant = 145, 35%, N strict = 145, 31%), and high sEAdi area under the baseline (N tolerant = 94, 23%, N strict = 79, 17%). Strict automated assessment allowed to reduce CoV of NMCdi to 15% from 37% for basic quality assessment. As PEEP was increased, NMCdi decreased significantly by 4.9 percentage point per cmH 2 O. Conclusion Advanced signal analysis of both Pocc and sEAdi greatly facilitates automated and well-defined identification of high-quality waveforms. In the critically ill, this approach allowed to demonstrate a dynamic NMCdi (Pocc/sEAdi) decrease upon PEEP increments, emphasising that sEAdi-based assessment of respiratory effort should be related to PEEP dependent diaphragm function. This novel, non-invasive methodology forms an important methodological foundation for more robust, continuous, and comprehensive assessment of respiratory effort at the bedside.