The application of dynamic methods for determining the oxygen uptake rate (OUR) in continuous mammalian cell cultures frequently ignores contributions to the oxygen balance such as the oxygen content in the culture medium inlet and outlet and the oxygen transfer between the culture and the headspace through the culture surface. We develop a mathematical model that allows OUR determinatioh in mammalian cell chemostat cultures through a dynamic method, incorporating these neglected variables, as well as the application and validation of this model at two culture temperatures, 37 degrees C and 33 degrees C. The proposed model was compared with a model that only includes the OUR term (typical model). The standard error of the proposed model was less than that of the typical model, making the proposed model more accurate. The results showed that at 37 degrees C, the results significantly differed depending on which model was used. At 33 and 37 degrees C, the specific oxygen uptake rate (q(O2)) values obtained with the proposed model were within the range usually reported for mammalian cells. It was also shown that the OUR value was underestimated if the oxygen transfer through the headspace-medium interface was not considered under mild hypothermia conditions. The model could be applied for developing strategies based on OUR monitoring and controlling for recombinant protein production under conditions of mild hypothermia. (C) 2016 Elsevier Ltd. All rights reserved.