Respiration of heterotrophic microorganisms decomposing soil organic carbon releases carbon dioxide from soils to the atmosphere. In the short term, soil microbial respiration is strongly dependent on temperature. In the long term, the response of heterotrophic soil respiration to temperature is uncertain. However, following established evolutionary trade-offs, mass-specific respiration (Rmass) rates of heterotrophic soil microbes should decrease in response to sustained increases in temperature (and vice-versa). Using a laboratory microcosm approach, we tested the potential for the Rmass of the microbial biomass in six different soils to adapt to three, experimentally imposed, thermal regimes (constant 10, 20 or 30 °C). To determine Rmass rates of the heterotrophic soil microbial biomass across the temperature range of the imposed thermal regimes, we periodically assayed soil subsamples using similar approaches to those used in plant, animal and microbial thermal adaptation studies. As would be expected given trade-offs between maximum catalytic rates and the stability of the binding structure of enzymes, after 77 days of incubation Rmass rates across the range of assay temperatures were greatest for the 10 °C experimentally incubated soils and lowest for the 30 °C soils, with the 20 °C incubated soils intermediate. The relative magnitude of the difference in Rmass rates between the different incubation temperature treatments was unaffected by assay temperature, suggesting that maximum activities and not Q10 were the characteristics involved in thermal adaptation. The time taken for changes in Rmass to manifest (77 days) suggests they likely resulted from population or species shifts during the experimental incubations; we discuss alternate mechanistic explanations for those results we observed. A future research priority is to evaluate the role that thermal adaptation plays in regulating heterotrophic respiration rates from field soils in response to changing temperature, whether seasonally or through climate change.