Heat stress and excess dietary starch promote endotoxemia, hindering fat metabolism and productivity in dairy cows. For study 1, 10 non-pregnant lactating Holstein cows (273 ± 35 d in milk) were administered a single bolus of saline (3 mL of saline; n 5) or LPS (0.375 µg of LPS/kg of body weight; n 5). Simultaneously, cows were intravenously infused a triglyceride emulsion and feed restricted for 16 h to induce hyperlipidemia in an attempt to model the periparturient period. Plasma samples were analyzed using untargeted lipidomics and metabolomics. Endotoxin increased circulating serum amyloid A, LPS-binding protein, cortisol, and ceramide concentrations. Endotoxin administration decreased plasma lysophosphatidylcholine (LPC) concentrations and ratio of phosphatidylcholine to phosphatidylethanomanine. Endotoxin administration also increased plasma concentrations of pyruvic and lactic acids, and decreased plasma citric acid concentration. In conclusion, acute intravenous LPS administration decreased circulating LPC concentrations, modified ceramide and glycerophospholipid concentrations, and influenced intermediary metabolism in dairy cows experiencing hyperlipidemia. For study 2, 12 multiparous Holstein cows (83 ± 27 days in milk) were divided into two main plots 1) unsupplemented or 2) supplemented with natural betaine (100 g/d delivered by intraruminal bolus). Within each plot, cows were randomly assigned to 1) heat stress (HS), 2) HS with partial rumen content transplantation (25%) from ad-libitum fed cows in thermoneutrality (HSRT), and 3) pair-feeding in thermoneutrality (TNPF) in a Latin square design of 14-d periods with 7-d washouts. Milk yield and milk content yield were reduced in HS relative to TNPF. Plasma insulin and fecal calprotectin were increased, while plasma total fatty acids were decreased in HS, relative to TNPF. We did not see any significant difference between HSRT and HS regarding production, plasma variables and gut permeability. Betaine supplementation increased plasma betaine in supplemented cows, however we did not observe any significant changes regarding production and metabolic responses. For study 3, 74 multiparous Holstein dairy cows (229 ± 1.41 d of gestation) were enrolled at -54 ± 1.41, relative to calving and received common prepartum diet. Periparturient cows received diets unsupplemented (UI; n = 35) or supplemented (SI; n = 39) with an IFA at 227 g/d from -48 to 42 d, relative to expected calving. Postpartum cows were divided by dietary starch low (LS; 21%) vs. high (HS; 27%) and IFA supplementation: LSUI, n = 17; HSUI, n = 18; LSSI, n = 19; HSSI, n = 20. An I.V. LPS challenge was introduced at 0.0625 µg/kg of BW (E. coli; O111:B4) on d 21 ± 3, relative to calving for all treatment groups. For the duration of the postpartum evaluation and post-LPS challenge, different dietary starch levels had no significant impact on DMI or milk yields, however, DMI and milk yields were greater in UI, relative to SI. Plasma glucose concentrations were lower in LS, relative to HS while insulin levels were lower in UI, relative to SI. Plasma free cholesterol, β-hydroxybutyrate (BHB) and total bilirubin concentrations and steatosis score were greater while serum amyloid A concentrations were lower in LS, relative to HS. Within 24 h of LPS bolus, eosinophil counts, plasma haptoglobin concentrations were lower in LS, relative to HS. In addition, hematocrit, RBC and eosinophils counts, and serum IL-8 concentrations were lower in UI, relative to SI cows. We conclude that postpartum high dietary starch concentrations elevate circulating markers of inflammation and supplementation of IFA during transition period modified milk production and immune responses.