The authors conducted a three-dimensional computational fluid dynamics (CFD) simulation to calculate the flow field in the inverted frustoconical shaking bioreactor with 5 L working volume (IFSB-5L). The CFD models were established for the IFSB-5L at different operating conditions (different shaking speeds and filling volumes) and validated by comparison of the liquid height distribution in the agitated IFSB-5L. The "out of phase" operating conditions were characterized by analyzing the flow field in the IFSB-5L at different filling volumes and shaking speeds. The values of volumetric power consumption (P/VL ) and volumetric mass transfer coefficient (kL a) were determined from simulated and experimental results, respectively. Finally, the operating condition effect on P/VL and kL a was investigated. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:478-485, 2018.