Fibre fractionation in the Hydrodynamic Fractionation Device (HDF) was studied for changing suspension flow parameters, i.e. different channel Reynolds numbers Re and accept flow rates up to 20% of the feed flow rate. The suspension flow behaviour was described using images recorded with a high-speed camera system. Fractionation performance was determined based on mass balances for a variety of length fractions of the pulp. Low Reynolds number flow characterised by Re = 1300 led to the formation of a fluid gap between the wall and the fibres located at the channel centre. Best fractionation performance was achieved for flow at this Reynolds number: no fiber removal was observed at 10% accept flow rate, and only 1% of the fibres were removed at 20% accept flow rate. A design space was established that highlights the optimum settings for fractionation in an HDF, which at low Re and high accept flow rate. Surprisingly, we found a significant increase of fines mass flow rate in the accept upon an increase of the Reynolds number. We speculate that a flow regime-dependent interaction of fines with the fibres exists in the HDF that critically affects the amount of fines in the fluid gap near the wall.