Our study aimed to assess the influence of the design of shake flask experiments on the growth of At. ferrooxidans using elemental sulfur (S0) as substrate. Cultures of At. ferrooxidans in 0Km medium with 1% sulfur were incubated at 30 °C for 10 days. Different culture volumes (75–150 mL), agitation rates (101–350 rpm), and orbital diameters (19–25 mm) were used to vary the gas–liquid mass transfer coefficient (kLa, from 8 to 41 h−1) and the power dissipation per unit of volume (P0/V, from 21 to 982 W/m3) from one culture to another. At. ferrooxidans growth and S0-biooxidation rate were improved with the increase of kLa and P0/V. In all experiments, dissolved oxygen concentration remained above 85% of saturation, indicating that there was no limitation of the gas–liquid mass transfer. Increasing P0/V for a given value of kLa improved the growth rate while it remained the same for a fixed P0/V and different kLa. It was also observed that μmax reached a plateau when P0/V was above 600 W/m3. Interestingly, at the plateau, the agitation rate was higher than the theoretical critical rate Nc required to achieve a complete suspension of the sulfur particles, which was not the case for the other cultures. These results show that microbial growth of At. ferrooxidans on S0 was influenced by particle dispersion and not by gas–liquid mass transfer. In this case, P0/V and Nc are two key factors that must be considered in the design of the experiments.