This study investigates the effect of temperature (37 °C, 55 °C and 70 °C) and inoculum-to-substrate ratio (ISR) (0.25, 0.5, and 1) on biochemical hydrogen potential (BHP) of 1G feedstock, sugar syrup through dark fermentation using mixed anaerobic consortia. The highest cumulative hydrogen yield of 188.85 NL-H2/kg-VSadded was obtained at 37 °C and ISR of 1 followed by 149.97 NL-H2/kg-VSadded at 55 °C and ISR of 0.5. Least hydrogen yield was obtained at 70 °C with the maximum of 20.84 NL-H2/kg-VSadded at ISR 1. Cone Model was the best fit for the BHP assysses at all the ISRs at 37 °C and 70 °C with R2 value near to 1 and the RMSE value less than 1. However, at 55 °C, First-Order and Modified Gomertz Model fitted best at different BHP ISRs. Hydrogen production occurred via both acetic acid and butyric acid pathways at 37 °C, dominated by Clostridium butyricum. The presence of fermentative and N2-fixing Proteobacteria at 37 °C and 55 °C also enhanced the respective biohydrogen yields. Thermoanaerobacter spp. dominated at 70 °C, producing a high concentration of acetic acid. Inhibition of microbial activity by an excessive level of acetic acid for a prolonged period led to the lowest biohydrogen yield at 70 °C.