biostudies-literatureFreitas DIrish Department of Agriculture and MarineUniversity of SurreyEuropean Union's Horizon 2020 ERA-NET co-fund ERA-HDHLDutch Ministry of Agriculture, Nature and Food QualityIndependent Fund Denmark - Danmarks Frie ForskningsfondEuropean Union’s Horizon 2020 research and innovation program Marie Sklodowska-Curie actionsResearch IrelandEuropean Union’s Horizon 2020 ERA-NET co-fund ERA-HDHLResearch Ireland (previously Science Foundation Ireland and the Irish Research Council)Norwegian Fund for Research Fees for Agricultural ProductsEuropean Union's Horizon 2020 research and innovation program Marie Sklodowska-Curie actionsResearch Foundation FlandersBBSRC Institute Strategic Programme Food Microbiome and Health30985https://www.ebi.ac.uk/biostudies/studies/S-EPMC12373966biostudies-literatureUnknown15(1)The activity of α-amylases is frequently determined using a single-point assay at 20 °C. Previous work within INFOGEST "Working Group 5 - Starch digestion and amylases" identified significant interlaboratory variation with this protocol. The current study aimed to evaluate the repeatability (intralaboratory precision) and reproducibility (interlaboratory precision), measured as coefficients of variation (CVs), of a newly optimized protocol version based on four time-point measurements at 37 °C. Human saliva (a pool from ten healthy adults) and three porcine enzyme preparations (two pancreatic α-amylases and pancreatin) were tested in 13 laboratories across 12 countries and 3 continents. Assay repeatability for each lab remained below 20% for all test products and the overall repeatability Scientific reportsInterlaboratory validation of an optimized protocol for measuring α-amylase activity by the INFOGEST international research network.PMC12373966KB-37-001-00712A0225N101107486BB/X011054/121/PATH-S/9418NRC 314599GA N° 6962958980131051-00046BCorbin KRSimsek SHenry GVidal NPPerez-Moral NLazaridou AAlongi MGwala SAriens RMCOrfila CWheller FGrassby TZhang BBoesch CBallance SEl SNLe Feunteun SLopez-Rodulfo IMKrause SKarakaya SKotsiou KDuijsens DFreitas DHafiz MSTomassen MMMMartinez MMBastiaan-Net SfalseInterlaboratory validation of an optimized protocol for measuring α-amylase activity by the INFOGEST international research network.The activity of α-amylases is frequently determined using a single-point assay at 20 °C. Previous work within INFOGEST "Working Group 5 - Starch digestion and amylases" identified significant interlaboratory variation with this protocol. The current study aimed to evaluate the repeatability (intralaboratory precision) and reproducibility (interlaboratory precision), measured as coefficients of variation (CVs), of a newly optimized protocol version based on four time-point measurements at 37 °C. Human saliva (a pool from ten healthy adults) and three porcine enzyme preparations (two pancreatic α-amylases and pancreatin) were tested in 13 laboratories across 12 countries and 3 continents. Assay repeatability for each lab remained below 20% for all test products and the overall repeatability 2025-01-01T00:00:00Z2025 Aug2026-05-08T06:50:44.161Z2026-04-07T23:30:51.129ZS-EPMC123739664084671410.1038/s41598-025-12561-y