Sludge treatment reed bed (STRB) systems combine dewatering, stabilisation and long-term storage of sludge. The main objective of this study was to investigate how substance concentrations change in the sludge residue during treatment and to conduct substance flow analyses covering the flow of substances in an STRB system over a 12-year treatment period, followed by three months' post-treatment in a stockpile area (SPA). Samples of sludge, reject water and sludge residue of different ages were collected at two Danish STRB system facilities and analysed for content of relevant substances. Concentrations of carbon and nitrogen in the sludge residue residing in an STRB system changed as a function of treatment time, mainly due to mineralisation; only a negligible part was lost to reject water. Considering metals and phosphorus, the main share was accumulated in the sludge residue; only minor fractions were lost to mineralisation or reject water. Post-treatment in an SPA resulted in an increase in dry matter content from 24% to 32%. After treatment, the concentrations of heavy metals (lead, cadmium, nickel, zinc, copper and chromium) in the sludge residue met the threshold values stated by the Danish Environmental Protection Agency and the EU.