Introduction International resuscitation guidelines suggest to use positive end-expiratory pressure (PEEP) during manual ventilation of neonates. Aim of our study was to test the reliability of self-inflating bags (SIB) with single-use PEEP valves regarding PEEP delivery and the effect of different peak inflation pressures (PIP) and ventilation rates (VR) on the delivered PEEP. Methods Ten new single-use PEEP valves from 5 manufacturers were tested by ventilating an intubated 1kg neonatal manikin containing a lung model with a SIB that was actuated by an electromechanical plunger device. Standard settings: PIP 20cmH2O, VR 60/min, flow 8L/min. PEEP settings of 5 and 10cmH2O were studied. A second test was conducted with settings of PIP 40cmH2O and VR 40/min. The delivered PEEP was measured by a respiratory function monitor (CO2SMO+). Results Valves from one manufacturer delivered no relevant PEEP and were excluded. The remaining valves showed a continuous decay of the delivered pressure during expiration. The median (25th and 75th percentile) delivered PEEP with standard settings was 3.4(2.7–3.8)cmH2O when set to 5cmH2O and 6.1(4.9–7.1)cmH2O when set to 10cmH2O. Increasing the PIP from 20 to 40 cmH2O led to a median (25th and 75th percentile) decrease in PEEP to 2.3(1.8–2.7)cmH2O and 4.3(3.2–4.8)cmH2O; changing VR from 60 to 40/min led to a PEEP decrease to 2.8(2.1–3.3)cmH2O and 5.0(3.5–6.2)cmH2O for both PEEP settings. Conclusion Single-use PEEP valves do not reliably deliver the set PEEP. PIP and VR have an effect on the delivered PEEP. Operators should be aware of these limitations when manually ventilating neonates.