Study objective was to determine effects of supplementing sucrose and lactose, with varying starch and rumen degradable protein (RDP) concentrations, on ruminal in vitro pH, volatile fatty acids (VFA), and forage neutral detergent fiber digestibility (ivFNDFD). The in vitro substrate was a concentrate mixture formulated for (DM basis) 21, 25 or 29% starch, 0, 2.5 or 5% of each sucrose and lactose and 10 or 12% RDP (3 × 3 x 3 × 2 factorial), placed in the media, and a 70:30 corn silage: alfalfa haylage mixture placed in an Ankom F57 bag and then the media. Two ruminally-cannulated lactating Holstein cows were fed a diet with (DM basis) 25% starch, 2.5% sucrose and 2.5% lactose to adapt the microbial population. The ivFNDFD procedure was the Combs-Goeser Assay with incubations of 24, 30 and 48 h. Each treatment was replicated 3 times and data were analyzed using PROC MIXED in SAS 9.4; model included fixed effects of RDP, starch, sucrose, lactose and incubation time, and the random effect of run. No differences were observed for pH, or effects of starch, RDP or sugar concentrations on ivFNDFD, but there was a tendency (P = 0.08) for a sucrose × lactose interaction, suggesting that sucrose and lactose may affect fiber digestibility differently. Increasing starch decreased acetate molar % (55.3, 54.4 and 53.4 ± 0.84% for 21, 25 and 29% starch, respectively; P < 0.0001) and increasing sucrose decreased acetate molar % (55.0, 54.8 and 54.1 ± 1.2% for 0, 2.5 and 5% sucrose, respectively; P = 0.003), but lactose had no effect. Starch increased butyrate molar % (14.1, 14.7 and 15.6 ± 0.40% for 21, 25 and 29% starch, respectively; P < 0.0001). Sucrose increased butyrate molar % (14.1, 14.7 and 15.2 ± 0.40% for 0, 2.5, and 5% sucrose, respectively; P = 0.0006), but lactose had no effect. Effects on molar proportions of acetate and butyrate in response to increasing starch and sucrose, but not lactose, suggests that sucrose and lactose may affect the ruminal fermentation differently. These results warrant further research exploring the potential interactions between sucrose and lactose in dairy rations.