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Biohydrogen production from buffalo slurry (BS) co-fermented with cheese whey (CW) and crude glycerol (CG) was investigated using a suitable microbial community (F210) as inoculum. Mixture Design was used to find the optimal composition (%) of the three substrate components and to investigate the effect of the mixing ratio on Bio-H₂ yields. Maximum H₂ yield estimated through the model was around 117 mL H₂/g VS_added, while the maximum experimentally detected was 111.6 ± 21.8 mL H₂/g VS_added, obtained for a mixing ratio of substrate composition of 66% BS and 33% CW (R² = 0.962; p-value = 0.0001). CW was clearly the most suitable substrate (with a relative contribution higher than 46%), but led to a rapid drop in pH from 6.5 to 4, while BS showed high buffering capacity by maintaining the pH above 6. Interestingly, the co-digestion of the different substrates decreased the H₂ production lag phase …