Abstract The development of the MicroResp™ approach ( Campbell et al., 2003 ) has allowed large-scale monitoring of community-level physiological profiles (CLPP). Here, we tested the sensitivity of this method to a carbon (C) substrate concentration gradient on 12 arable soils which had received contrasting long-term (53 years) managements. Irrespectively to the soil organic carbon (SOC), total activity and catabolic evenness were similar for C substrate addition above 10% C added of SOC, suggesting microbial respiratory metabolism saturation. Below this threshold, CLPP were significantly altered, especially when the amount of SOC was low. This threshold corresponded to 4 up to 10 mgC mL −1 soil water which is 66–87% lower than used in the original approach. Such C concentrations could be used in future MicroResp™ assaying when determining CLPP via multi-substrate induced respiration of the microbial biomass.