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Abstract Background Several factors may decrease the accuracy of quantitative PET myocardial perfusion imaging (MPI). It is therefore essential to ensure that myocardial blood flow (MBF) values are reproducible and accurate, and to design systematic protocols to achieve this. Until now, no systematic phantom protocols have been available to assess the technical factors affecting measurement accuracy and reproducibility in MPI. Materials and methods We implemented a standard measurement protocol, which applies a flow phantom in order to compare image-derived flow values with respect to a ground truth flow value with [15O]H2O MPI performed on both a Discovery MI (DMI-20, GE Healthcare) and a Biograph Vision 600 (Vision-600, Siemens Healthineers) system. Both systems have automatic [15O]H2O radio water generators (Hidex Oy) individually installed, allowing us to also study the differences occurring due to two different bolus delivery systems. To investigate the technical factors contributing to the modelled flow values, we extracted the [15O]H2O bolus profiles, the flow values from the kinetic modeling (Qin and Qout), and finally calculated their differences between test-retest measurements on both systems. Results The measurements performed on the DMI-20 system produced Qin and Qout values corresponging to each other as well as to the reference flow value across all test-retest measurements. The repeatability differences on DMI-20 were 2.1% ± 2.6% and 3.3% ± 4.1% for Qin and Qout, respectively. On Vision-600 they were 10% ± 8.4% and 11% ± 10% for Qin and Qout, respectively. The measurements performed on the Vision-600 system showed more variation between Qin and Qout values across test-retest measurements and exceeded 15% difference in 7/24 of the measurements. Conclusions A preliminary protocol for measuring the accuracy and reproducibility of flow values in [15O]H2O MPI between digital PET/CT systems was assessed. The test-retest reproducibility falls below 15% in majority of the measurements conducted between two individual injector systems and two digital PET/CT systems. This study highlights the importance of implementing a standardized bolus injection and delivery protocol and importance of assessing technical factors affecting flow value reproducibility, which should be carefully investigated in a multi-center setting.