Syngas fermentation has gained momentum over the last decades. The cost-efficient design of industrial-scale bioprocesses is highly dependent on quantitative microbial growth data. Kinetic and stoichiometric models for syngas-converting microbes exist, but accurate experimental validation of the derived parameters is lacking. Here, we describe a novel experimental approach for measuring substrate uptake kinetics of gas-fermenting microbes using the model microorganism Clostridium autoethanogenum. One-hour disturbances of a steady-state chemostat bioreactor with increased CO partial pressures (up to 1.2 bar) allowed for measurement of biomass-specific CO uptake- and CO₂ production rates ((Formula presented.), (Formula presented.)) using off-gas analysis. At a p_CO of 1.2 bar, a (Formula presented.) of −119 ± 1 mmol g⁻¹_X h⁻¹ was measured. This value is 1.8–3.5-fold higher than previously reported experimental and kinetic modeling results for syngas fermenters. Analysis of the catabolic flux distribution reveals a metabolic shift towards ethanol production at the expense of acetate at p_CO (Formula presented.) 0.6 atm, likely to be mediated by acetate availability and cellular redox state. We characterized this metabolic shift as acetogenic overflow metabolism. These results provide key mechanistic understanding of the factors steering the product spectrum of CO fermentation in C. autoethanogenum and emphasize the importance of dedicated experimental validation of kinetic parameters.