The objective of this study was to examine the interaction between partial replacement of dietary starch by digestible NDF (dNDF) and the speed of agitation on digestion and fermentation profile in vitro. Ten dual-flow continuous culture fermentors were utilized in a randomized complete block design experiment with treatments assigned in factorial arrangement: High starch diet (HS) with 29.9% starch and 13.5% dNFD (DM basis) vs. High dNDF diet (HF) with 22.0% starch and 18.6% dNDF, and agitation speeds of 75 vs. 125 rpm. Fermentors were blocked by period, with two 10-d periods (7-d adaptation and 3-d collection) being executed (n = 5). Liquid and solid dilution rates were set at 8 and 4%/h, respectively. Fermentors were fed once daily (0.023 mg DM/mL of buffer infused/d) and pH controlled to stay within a range of 5.5 to 7.0. No interactions were observed between agitation speed and dietary dNDF level for NDF and DM digestibilities or VFA concentration. Compared with 125 rpm, 75 rpm increased average pH (6.53 vs. 6.40; P = 0.01) and valeric acid concentration (4.73 vs. 3.21 mM; P ≤ 0.05) and tended to increase isovaleric acid concentration (3.50 vs. 2.43 mM; P = 0.09), without affecting any other parameters. Partial replacement of starch by dNDF increased NDF digestibility (43.2 vs. 35.5%; P = 0.03) and tended to decrease DM digestibility (64.0 vs. 61.7%; P = 0.10). Concentrations of ammonia (1.55 vs. 1.38 mg/dL), total VFA (60.2 vs. 47.1 mM) and butyric acid (12.8 vs. 9.89 mM) were higher in HF compared with HS (all P < 0.05). HF also tended to increase acetic (24.2 vs. 19.6 mM), propionic (13.7 vs. 10.5 mM), and isovaleric (3.32 vs. 2.56 mM) concentration (all P < 0.10). Although HF increased total VFA, no differences in average pH (6.44 vs. 6.48; P = 0.27) or time below 5.7 (130 vs. 158 min/d; P = 0.49) were observed, suggesting a slower rate of fermentation with HF compared with HS. In conclusion, speed of agitation did not affect most of the fermentation parameters evaluated and substitution of starch for dNDF increased NDF degradation and ammonia concentration.