The aim of this study was to investigate thermophilic (55 degrees C) aerobic digestion (TAD) as pre- and inter-stage treatment of sludge anaerobic digestion and to analyse the change in organic matter accessibility and complexity. Pre-treatment decreased methane yield (up to -70%), due to oxidation losses whereas inter-stage treatment slightly improved overall methane yield (+ 2.6%) and total COD removal (+ 5%) compared to control. Anaerobic degradability and COD removal in the second anaerobic stage significantly increased, by 13-40%. Organic matter fractionation showed that TAD led to an increase in sludge organic matter accessibility in all cases. Organic matter complexity, measured by fluorimetry, increased after TAD pre-treatment whereas it remained constant after inter-stage treatment. TAD was shown to be more efficient if applied to a more recalcitrant substrate and should thus be used as inter-stage treatment to avoid decreasing methane production.