BackgroundBoth one‐stage (OSA) and chromogenic substrate assays (CSA) are used to measure factor VIII (FVIII) activity. Factors explaining analytical variation in FVIII activity levels are still to be completely elucidated.AimThe aim of this study was to investigate and quantify the analytical variation in OSA and CSA.MethodsFactors determining analytical variation were studied in sixteen lyophilized plasma samples (FVIII activity <0.01‐1.94 IU/mL) and distributed by the ECAT surveys. To elucidate the causes of OSA variation, we exchanged deficient plasma between three company set‐ups.ResultsOn average, 206 (range 164‐230) laboratories used the OSA to measure FVIII activity and 30 (range 12‐51) used CSA. The coefficient of variation of OSA and CSA increased with lower FVIII levels (FVIII <0.05 IU/mL). This resulted in misclassification of a severe haemophilia A sample into a moderate or mild haemophilia A sample in 4/30 (13.3%) of CSA measurements, while this was 37/139 (26.6%) for OSA. OSA measurements performed with reagents and equipment from Werfen showed slightly lower FVIII activity (0.93, IQR 0.88‐0.98 IU/mL) compared to measurements with Stago (1.07, IQR 1.02‐1.14 IU/mL) and Siemens (1.03, IQR 0.97‐1.07 IU/mL). Part of this difference is explained by the value of the calibrator. For CSA, the measured FVIII levels were similar using the different kits.ConclusionsIn the lower range (<0.05 IU/mL), analytical variation of FVIII measurements is high in both OSA and CSA measurements. The variation in FVIII activity levels was partly explained by specific manufacturers. Further standardization of FVIII measurements and understanding of analytical variation is required.