In this work, the effects of various operating parameters of the two-stage yeast treatment on the VHG fermentation performance were evaluated. First, a central composite design was used to investigate the effects of temperature and pH on cell viability and flocculation dispersion in the acid treatment unit (1st stage). Second, the effects of temperature, oxidation-reduction potential (ORP) and sugar concentration in the yeast reactivation (2nd stage) on the performance parameters (productivity, ethanol yield and cell viability) of the VHG fermentation were evaluated through a full factorial design. The results indicated that the acid treatment around 2.65 pH and temperature controlled at 30 °C for 30 min led to higher cell viability. The conditions of yeast reactivation stage that positively influenced the VHG fermentation performance were relatively low temperature and negative ORP. A mechanistic model quantitatively revealed the influence of the operating parameters studied on cell growth kinetics. The two-stage yeast treatment promoted the increase in cell viability through the cell membrane recovery, which led to the maintenance or improvement the VHG fermentation performance.