Abstract The thermophilic anaerobic digestion of cheese whey was evaluated using a single and two stage configuration (H2–CH4) in a sequencing batch reactor (SBR). The single stage process presented stable performance with a specific methane production (SMP) of 314.5 ± 6.6 L CH4 kg−1 CODfeed (Chemical oxygen demand) at a hydraulic retention time (HRT) of 8.3 days. On the contrary, the two stage process presented instabilities at an HRT of 12.5 days with acid accumulation being observed in the methanogenesis phase at an earlier stage. This behaviour was indicative of process inhibition by high concentrations of sodium and potassium ions as a consequence of pH control during the H2 producing stage. In spite of this phenomenon, this condition attained the highest SMP value (340.4 ± 40 L CH4 kg−1 CODfeed). The performance of the anaerobic digestion process was also analysed by means of Fourier transform infrared (FTIR) and 1H nuclear magnetic resonance (1H NMR) spectroscopy. The two stage process showed higher content in triacylglycerol groups probably associated with changes in archaeal lipid complexes as a microbial response to a higher salinity environment.