Multiple-breath nitrogen washout (MBNW) and its clinical parameter lung clearance index (LCI) are gaining increasing attention for the assessment of small airway function. Measurement of LCI relies on accurate assessment of functional residual capacity (FRC). The EasyOne Pro LAB (ndd) and Exhalyzer D (EM) are two commercially available MBNW devices. The aim of the study was to compare these two devices in vitro and in vivo in healthy subjects with regard to FRC, LCI and secondary outcome parameters and to relate FRC_MBNW to FRC measured by body plethysmography (pleth) and helium dilution technique. MBNW measurements were performed using a lung model (FRC between 500 and 4000 mL) in vitro and in 38 subjects aged 6–65 years followed by helium dilution and pleth in vivo using fixed and relaxed breathing techniques. In vitro accuracy within 5% of lung model FRC was 67.3% for ndd, FRC was >5% higher for EM in all tests. In vivo, FRC_pleth ranged from 1.2 to 5.6 L. Mean differences (limits of agreement) between FRC_pleth and FRC_MBNW were −7.0%, (−23.2 to 9.2%) and 5.7% (−11.2 to 22.6%) using ndd and EM, respectively. FRC_ndd was consistently lower than FRC_EM (−11.8% (−25.6 to 2%)). LCI was comparable between the two devices (−1.3% (−21.9 to 19.3%)). There was a difference of >10 % in LCI in 12 of 38 subjects. Using the most recent software updates, both devices show relevant deviations in FRC measurement both in vitro and in vivo and individual differences in LCI in a significant proportion of subjects. The devices are therefore not interchangeable.