Aeration is an energy-intensive process of aerobic biological treatment in wastewater treatment plants (WWTP). Two-stage processes enable energy-efficient operation, but oxygen transfer has not been studied in depth before. In this study, α-factors were determined with long-term ex situ steady-state off-gas measurements in pilot-scale test reactors (5.8 m height, 8.3 m3) coupled to full-scale activated sludge basins. A two-stage WWTP with more than 1 Mio population equivalent was studied over 13 months including rain and dry weather conditions. Operating data, surfactant concentrations throughout the two-stage process, and the effect of reverse flexing on pressure loss of diffusers were examined. The values of αmean, αmin, and αmax for design load cases of aeration systems were determined as 0.45, 0.33, and 0.54 in the first high-rate carbon removal stage and as 0.80, 0.69, and 0.91 in the second nitrification stage, respectively. The first stage is characterized by a distinct diurnal variation and decrease in α-factor during stormwater treatment. Surfactants and the majority of the total organic carbon (TOC) load are effectively removed in the first stage; hence, α-factors in the second stage are higher and have a more consistent diurnal pattern. Proposed α-factors enable more accurate aeration system design of two-stage WWTPs. Fouling-induced diffuser pressure loss can be restored effectively with reverse flexing in both treatment stages.