Stirred tank bioreactors using suspension adapted mammalian cells are typically used for the production of complex therapeutic proteins. The hydrodynamic conditions experienced by cells within this environment have been shown to directly impact growth, productivity, and product quality and therefore an improved understanding of the cellular response is critical. Here we investigate the sub-lethal effects of different aeration strategies on Chinese hamster ovary cells during monoclonal antibody production. Two gas delivery systems were employed to study the presence and absence of the air–liquid interface: bubbled direct gas sparging and a non-bubbled diffusive silicone membrane system. Additionally, the effect of higher gas flow rate in the sparged bioreactor was examined. Both aeration systems were run using chemically defined media with and without the shear protectant Pluronic F-68 (PF-68). Cells were unable to grow with direct gas sparging without PF-68; however, when a silicone membrane aeration system was implemented growth was comparable to the sparged bioreactor with PF-68, indicating the necessity of shear protectants in the presence of bubbles. The cultures exposed to increased hydrodynamic stress were shown by flow cytometry to have decreased F-actin intensity within the cytoskeleton and enter apoptosis earlier. This indicates that these conditions elicit a sub-lethal physiological change in cells that would not be detected by the at-line assays which are normally implemented during cell culture. These physiological changes only result in a difference in continuous centrifugation performance under high flow rate conditions. Product quality was more strongly affected by culture age than the hydrodynamic conditions tested. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013.