A strategy was evaluated for conditioning activated sludge biomass to a new substrate whereby the polyhydroxyalkanoate (PHA) accumulation capacity of the biomass was enhanced based on a series of aerobic feast–famine acclimation cycles applied prior to PHA accumulation. Different biomass types enriched during the treatment of municipal wastewater at laboratory, pilot, and full scales were exposed to aerobic feast–famine acclimation cycles at different feast-to-famine ratios with an acetate–propionate mixture (laboratory scale), acetate (pilot scale), and fermented waste–sludge centrate (pilot scale). A sevenfold increase in specific PHA storage rates and 20% increase in substrate utilization rates were observed during acclimation cycles (laboratory acetate–propionate). Biomass acclimation led to more than doubling of the specific substrate utilization rates, PHA storage rates, biomass PHA contents, and specific PHA productivities (per initial biomass) during PHA accumulation. The biomass PHA contents were found to increase due to acclimation from 0.19 to 0.34 (laboratory acetate–propionate), 0.39 to 0.46 (pilot acetate) and 0.19 to 0.25 gPHA/gVSS (pilot centrate). A similar bacterial community structure during acclimation indicated that a physiological rather than a genotypic adaptation occurred in the biomass. The physiological state of the biomass at the start of PHA accumulation was deemed significant in the subsequent PHA-accumulation performance. Positive acclimation trends can be monitored by measuring the relative increase in feast substrate utilization or respiration rates with respect to those of the first acclimation cycle.