Functional residual capacity (FRC) accuracy is essential for deriving multiple-breath nitrogen washout (MBNW) indices, and is the basis for device validation. Few studies have compared existing MBNW devices. We evaluated in vitro and in vivo FRC using two commercial MBNW devices, the Exhalyzer D (EM) and the EasyOne Pro LAB (ndd), and an in-house device (Woolcock in-house device, WIMR).FRC measurements were performed using a novel syringe-based lung model and in adults (20 healthy and nine with asthma), followed by plethysmography (FRC_pleth). The data were analysed using device-specific software. Following the results seen with ndd, we also compared its standard clinical software (ndd v.2.00) with a recent upgrade (ndd v.2.01).WIMR and EM fulfilled formal in vitro FRC validation recommendations (>95% of FRC within 5% of known volume). Ndd v.2.00 underestimated in vitro FRC by >20%. Reanalysis using ndd v.2.01 reduced this to 11%, with 36% of measurements ≤5%. In vivo differences from FRC_pleth (mean±sd) were 4.4±13.1%, 3.3±11.8%, −20.6±11% (p<0.0001) and −10.5±10.9% (p=0.005) using WIMR, EM, ndd v.2.00 and ndd v.2.01, respectively.Direct device comparison highlighted important differences in measurement accuracy. FRC discrepancies between devices were larger in vivo, compared to in vitro results; however, the pattern of difference was similar. These results represent progress in ongoing standardisation efforts.