The fluid dynamics of hollow fibre bioreactors was studied using residence time distribution (RTD) and protein polarisation experiments. Small differences in the densities of tracer protein solution and carrier liquid resulted in broad RTDs with long tails due to settling of tracer solution near the upstream extracapillary space (ECS) port. Moreover, module orientation and ECS flow direction had a significant influence on the observed RTDs. After matching the densities of the tracer and the carrier fluid, the tracer moved through the ECS as a dispersed plug. The reactor orientation and flow direction did not influence RTDs in these density-matched experiments. The reactor orientation had a dramatic effect on protein polarisation in the ECS.