An experimental study was conducted to describe mass transfer impacts within nitrifying aggregates sourced from sequencing batch reactor (SBR) activated sludge systems. Flocculent and granular sludge with high nitrification activity was obtained in two laboratory SBR systems, supplied with a synthetic, ammonium-based feed. The flocculent biomass was fractionated using a sieving procedure, in order to obtain biomass fractions with different particle size distributions. The oxygen uptake rate (OUR) response to changes in dissolved oxygen concentration was measured under highly controlled conditions in a titrimetric and off-gas analysis (TOGA) sensor, and the results used to assess mass transfer effects. As the average particle size of the biomass increased, mass transfer limitations were found to increase significantly. Empirically fitted, apparent K(S,O2) values were demonstrated to be highly dependent on particle size, and reflect the mass transfer limitations occurring in the aggregates within a given system. Such parameters thus have little to do with the actual biokinetic parameter from which they are derived. The results obtained from the TOGA sensor study were consistent with those obtained from a microelectrode study on the same nitrifying granules. Together, these studies add considerable weight to the conclusion that consideration of external and internal mass transfer limitations is vital to the accurate description of activated sludge treatment processes, particularly those with a high oxygen uptake rate.