Abstract Hydrogen concentration is important for the metabolic distribution in mixed culture fermentation (MCF) but hydrogen supersaturation is often ignored. In this study, hydrogen supersaturation in thermophilic MCF was investigated online by a membrane inlet mass spectrometry. The results showed that with the increase of glucose loading rate (from 13.5 to 137.5 mmol/L/d) and the decrease of Reynolds number (from 12,900 to 3500), the hydrogen partial pressure ( P H 2 ) remained almost unchanged, but the hydrogen concentration in liquid (H2aq) increased from 0.82 to 1.27 and from 0.68 to 1.21 mmol/L, respectively. It demonstrated that hydrogen supersaturation occurred and the supersaturation ratio was between 1.7 and 3.0. Meanwhile, higher H2aq resulted in lower hydrogen yield, lower glucose degradation rate and higher mole ratio of ethanol/(acetate + butyrate). Thus, H2aq is more appropriate than P H 2 when discussing the H2 role in MCF. Furthermore, the calculated KLa clearly illustrated that the required KLa values for maintaining low H2aq were order of magnitudes higher than the experimental ones. Therefore, hydrogen supersaturation is inevitable in practice and should be considered in MCF.