The current study was designed to investigate the effect of feeding diets with or without soybean oil and the replacement of a portion of the starch with high sugar (Su) and high soluble fiber (SF) on in vitro fermentation. We hypothesized that high Su or high SF would improve fermentation parameters in continuous cultures when challenged with soybean oil and high starch. An experiment was conducted using 8 dual-flow continuous culture fermentors. Treatments included soybean oil (High PUFA; HF) or no soybean oil (Low PUFA; LF), low (LSu; 4% DM) or high sugar (HSu; 9% DM), and low or high soluble fiber (SF; 6 or 12% DM). This resulted in 8 unique treatments, 2 PUFA levels: LF (2.5% EE) and HF (5% EE) and to a combination of Su and SF sequence [LSuLSF (4% Su, 6% SF); LSuHSF (4% Su, 12% SF); HSuLSF (9% Su, 6% SF); and HSuHSF (9% Su, 12% SF)] within PUFA level administered according to a split-plot, 4 × 4 Latin square design. Cultures were run for 4 periods of 10 d, with fresh inoculum used at the start of each period. Statistical analysis was conducted using the MIXED procedure of SAS. High PUFA-fed fermentors had lower digestibility coefficients for NDF, ADF, DM, and OM (P = 0.01), but did not alter VFA production or pH. Mean culture pH was higher (P < 0.01) in the HSu vs LSu-fed fermentors (6.14 vs. 5.95 ± 0.13). Mean pH was not altered by SF, but lactate tended (P = 0.06) to be lower for HSF versus LSF (9.35 vs. 9.61 ± 1.93). There was a tendency (P = 0.09) for total VFA to be increased with HSu treatments (P = 0.09). In addition, there was a significant reduction (P = 0.02) in propionate and an increase (P = 0.03) in butyrate concentrations with Su. Inclusion of SF exhibited an increase in the proportions of acetate (P < 0.01), and a reduction of propionate (P < 0.01).The results of this study imply that replacing a portion of the starch with sugar or SF may improve rumen fermentation.