In this study, the co-fermentation of carbohydrates and proteins at different ratios (C1-C5) was explored. The rates of particulate carbohydrates degradation in the co-substrate mixtures, not only increased with starch concentrations, but negatively impacted the degradation rates of the particulate proteins. Particulate proteins also negatively impacted particulate carbohydrate degradation rates, albeit to a lesser extent. Generally, there was a synergistic impact on hydrogen production and the optimum ratio that required no pH control occurred at C4 (80% carbohydrates + 20% proteins) with a hydrogen yield of 350 mL H-2/gCOD(added) which was 38% higher than the expected, and the fermentation followed the acetate-ethanol pathway. Response Surface Methodology (RSM) was used to optimize the responses to the co-fermentation process at C4. By fitting 20 experimental data points, the responses adequately fitted second-order polynomial models. At the optimized VFA and ammonia concentrations of 580 mg/L and 40 mg/L, respectively, the biohydrogen production process would be feasible without pH control at a carbohydrate-to-protein COD ratio of 4:1. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.