Microbiome analyses of soils and microcosm experiments depend on conditions that include sterilization in order to perform experimental manipulation of microbial communities. Still, they should represent conditions close to nature. When using metal contaminated soils, sterilization methods might alter metal availability. Here, four typical metal contaminated substrates were analyzed, representing different contamination histories and soil types. They included two very poor substrates, as they are often found at metal contaminated sites. The low contents in organic carbon and nitrogen as well as two substrates with slightly higher nutrient availability were used to perform a comprehesive study for element availability changes induced by sterilization. Autoclaving, dry heat or gamma ra gamma sterilization were applied and compared to a non-treated control. The sterile substrates were analyzed using sequential extraction to account for different associations of the elements. Metals forming specific (hydro)oxide layers were specifically analyzed since they in turn may also impact other metals or ions. In addition, (heavy) metals and (micro)nutrients were analyzed for changes in speciation. The effects of autoclaving (wet heat) was found acceptable, while gamma-ray irradiation did show unexpected changes in metal associations, especially for one substrate. Dry heat changed metal availability to the highest degree. (C) 2017 Elsevier B.V. All rights reserved.