Background: This study investigated the effect of organic loading rate (OLR) and NaCl concentration on biohydrogen production by preheated anaerobic sludge in a lab scale anaerobic sequencing batch reactor (ASBR) fed with glucose during long time operation. Methods: During ASBR operation, the OLR was increased in steps from 0.5 to 5 g glucose/L.d and NaCl addition started at an OLR of 5 g glucose/L.d, to obtain NaCl concentrations in the reactor in the range of 0.5-30 g/L. Results: With an increasing OLR from 0.5 to 5 g glucose/L.d, the biohydrogen yield increased and reached 0.8 ± 0.4 mol H2/mol glucose at an OLR of 5 g glucose/L.d. A NaCl concentration of 0.5 g/L resulted in a higher yield of biohydrogen (1.1 ± 0.2 mol H2/mol glucose). Concentrations above 0.5 g/L NaCl led to decreasing biohydrogen yield and the lowest yield (0.3 ± 0.1 mol H2/mol glucose) was obtained at 30 g/L of NaCl. The mass balance errors for C, H, and O in all constructed stoichiometric reactions were below 5%. Conclusions: The modified Monod model indicated that r (H2)max and Ccrit values were 23.3 mL H2/g VSS/h and 119.9 g/L, respectively. Additionally, ASBR operation at high concentrations of NaCl shifted the metabolic pathway from acidogenic toward solventogenic.