Scale-up of bioprocesses is hampered by open questions, mostly related to poor mixing and mass transfer limitations. Concentration gradients of substrate, carbon dioxide, and oxygen in time and space, especially in large-scale high-cell density fed-batch processes, are likely induced as the mixing time of the fermentor is usually longer than the relevant cellular reaction time. Cells in the fermentor are therefore repeatedly exposed to dynamic environments or perturbations. As a consequence, the heterogeneity in industrial practices often decreases either yield, titer, or productivity, or combinations thereof and increases by-product formation as compared to well-mixed small-scale bioreactors, which is summarized as scale-up effects. Identification of response mechanisms of the microorganism to various external perturbations is of great importance for pinpointing metabolic bottlenecks and targets for metabolic engineering. In this review, pulse response experimentation is proposed as an ideal way of obtaining kinetic information in combination with scale-down approaches for in-depth understanding of dynamic response mechanisms. As an emerging tool, computational fluid dynamics is able to draw a holistic picture of the fluid flow and concentration fields in the fermentor and finds its use in the optimization of fermentor design and process strategy. In the future, directed strain improvement and fermentor redesign are expected to largely depend on models, in which both microbial kinetics and fluid dynamics are thoroughly integrated.