Ever since the potential of inclusion bodies (IBs) has been recognized, substantial advances have been made towards understanding IB processes and enabling efficient and controlled development strategies. Still, the influence of the chosen upstream processing (USP) strategy on the properties of inclusion bodies (IBs) and their refolding performance remains poorly understood. This work aims to target this challenge by investigating the influence of two chosen USP parameters, namely the specific substrate uptake rate and the temperature during induction, on IB titer, IB properties, namely IB purity, size and secondary protein structure of the IBs, as well as refolding yield of single-chain variable fragment M (scFvM) IBs. Contrary to findings in the literature, USP conditions neither had a statistically significant effect on the aforementioned IB properties nor on the refolding yield, but could clearly alter the IB titer. Our results provide detailed analytical insights on the independence of IB properties from USP conditions for this protein, while increasing the volumetric IB productivity proved feasible through variations in USP parameters. Therefore, titer maximization appears to be the sole optimization strategy for scFvM IBs and these findings may also apply to other target proteins with similar structural properties.