The steady-state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI-TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP-Hex, UDP-HexNAc). By switching to a C-13(6) glucose containing feed media during constant operation at 20 x 10(6) cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the C-13(6) glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools, ST (0.56 days), which were confirmed in the bioreactor. On the other hand, it is shown that the reactor residence time R (1 day) and characteristic time for glucose uptake Glc (0.33 days), representative of the bioreactor dynamics, delayed the consumption of C-13(6) glucose in the bioreactor and thus the intracellular C-13 enrichment. The proposed experimental approach allowed the decoupling of bioreactor hydrodynamics and intrinsic dynamics of cell metabolism in response to a change in the cell culture environment. (c) 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1630-1639, 2017