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Description: We investigated if and how changes of MRI hardware, scanning protocols or preprocessing software affect diffusion-weighted imaging. We therefore compared DWI outcomes and artefact severity of 121 healthy participants (age range 19-54 years) who underwent two matched DWI protocols (Siemens product and Center for Magnetic Resonance Research sequence) at two sites (Siemens 3T Magnetom Verio and Skyrafit). We demonstrated that DTI outcome measures strongly depend on imaging site and software, and that these biases vary between brain regions. These regionally inhomogeneous biases may exceed and considerably confound physiological effects such as ageing, highlighting the need to harmonise data acquisition and analysis.