Grain induced subacute ruminal acidosis has been linked to systemic inflammation. We hypothesized that a rumen and hindgut starch challenge would stimulate an inflammatory response in intestinal tissue. Six rumen cannulated nonlactating nonpregnant Holstein cows were assigned according to a randomized block design to a control diet (CON) or CON top-dressed with 20% ground barley (STARCH). Diets were restricted fed to achieve weight gains of ~45 kg by the end of the 20-wk experiment. STARCH cows also received abomasal infusions of corn starch (4 g/kg BW per day) and CON cows received abomasal infusions of water pulse dosed twice a day during wk 8, 12, 16 and 19–20. Rumen fluid, feces and blood were collected weekly (non-infusion weeks) or 3 times weekly (infusion weeks). Rumen fluid and feces were analyzed for pH, lactate and VFA and blood samples were analyzed for haptoglobin (Hp) and serum amyloid A. At wk 20, cows were euthanized and mesenteric adipose, colon and jejunum were collected for determination of immune cell phenotype. Weekly data were analyzed using a repeated measures Glimmix model in SAS that included fixed effects of diet, month, infusion, and 2 way interactions and the random effect of cow. Immune cell phenotype data were analyzed with a model including effect of diet only. Diet × infusion tended to affect Hp (P = 0.08) and fecal pH (P = 0.10), lactate (P = 0.08) and butyrate (P = 0.09). Interactions were due to effects observed during infusion periods, when Hp and fecal pH tended to be lower in STARCH cows. Fecal lactate and butyrate tended to be higher in STARCH cows. Diet did not affect immune cell phenotype. Overall means (% total) of dendritic cell markers MHCII, CD40, CD80 and CD86 were 25.1%, 1.1%, 0.9%, 0.2% (adipose), 25.2%, 9.3%, 26.4%, 3.3% (colon) and 13.7%, 3.3%, 14.2%, 1.7% (jejunum), respectively. Means of effector T-cell markers CD4 and CD8 were 14.6%, 22.6% (colon) and 4.2%, 12.2% (jejunum), respectively. Although replication was low, the rumen and hindgut starch challenge did not appear to induce systemic inflammation or inflammatory cell infiltration into the intestines.