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The hydromechanical stress is a relevant parameter for mammalian cell cultivations, especially regarding scale-up processes. It describes the mechanical forces exerted on cells in a bioreactor. The maximum local energy dissipation rate is a suitable parameter to characterize hydromechanical stress. In literature, different studies deal with the effects of hydromechanical stress on CHO cells in stirred tank reactors. However, they often focus on lethal effects. Furthermore, systematic examinations in smaller scales like shake flasks are missing. Thus, this study systematically considers the influence of hydromechanical stress on CHO DP12 cells in shake flask cultivations. By utilizing online monitoring of the oxygen transfer rate, the study simplifies and enhances the resolution of examinations. Results indicate that while lethal effects are absent, numerous sub-lethal effects emerge with increasing hydromechanical stress: The process time is prolonged. The time of glucose and glutamine depletion, and the lactate switch correlate positively linear with the logarithmic average energy dissipation rate while the maximum specific growth rate correlates negatively. Strikingly, the final antibody concentration only declines at the highest tested average energy dissipation rate of 3.84 W kg -1 (only tested condition with a turbulent flow regime and therefore a higher maximal local energy dissipation rate) from about 250 mg L -1 to about 180 mg L -1 . This study presents a straightforward method to examine the impact of hydromechanical stress in shake flasks, easily applicable to any other suspension cell line. Additionally, it offers valuable insights for scale-up processes, for example into stirred tank reactors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)