Aiming at increasing productivity in mammalian cell processes, mild hypothermia and supplementation with short-chain fatty acids have been investigated in literature, but usually separately. The combined effects of butyric acid and hypothermia were investigated in a few studies, but despite the lower cytotoxicity of valeric acid no reports were found on its use under low temperatures. In this work, DOE was used to compare supplementation with both butyrate and valerate at 31, 34 and 37 ◦C to enhance recombinant protein productivity in a CHO cell process. Due to the promising results obtained with valerate, this fatty acid was further investigated. According to the results obtained, 1 mM valeric acid at 31 ◦C enables high cell viabilities, higher product titres and increases cell specific productivity (qp) by approximately 4-fold. Cell cycle analysis showed that at 31 ◦C, especially with valerate, a higher percentage of cells was in G0 + G1 phase. Overall, an inverse relation between qp and cell proliferation state [S/(G0 + G1) ratio] was observed. To our knowledge, this is the first work describing the effects of valeric acid under mild hypothermia on CHO cell cultivations. The results provide a promising tool to increase qp under lower proliferation rates, which can be very useful to develop robust high-cell-density, high-productivity perfusion processes.