Heifers (n = 4/genotype) from unselected (stable milk yield since 1964, UH) and contemporary (CH) Holsteins that differed in milk yield (6,200 and 11,100 kg milk/305 d) or from Red Angus cows (RA) were fed the same diet ad lib and housed together for 47 d before being challenged with 0.5 μg LPS/kg BW. Heifers were 20 mo old and pregnant except for 2 CH heifers that were synchronized to be at d 8 of their cycle at the first LPS challenge (C1). Progesterone exceeded 3.5 ng/mL at C1. Jugular catheters were implanted 24 h before LPS (Escherichia coli O111:B4). Blood samples were collected at −1, −0.5, 0, 1, 2, 3, 4, 6, 8, and 24 h relative to LPS administration and plasma harvested. Body temperatures (BT) were determined at these times and at 5 and 7 h. A second identical LPS challenge (C2) was administered 4 d later. Data were analyzed by repeated measures using PROC MIXED (SAS). Means differed when P < 0.05. Cortisol, interleukin-6 (IL-6), xanthine oxidase (XO), and tumor necrosis factor α (TNFα) were greater (P < 0.01) in C1 than C2, BT and IGF-1 were less (P < 0.01) and glucose and nitric oxide (NOX) did not differ (P > 0.11) between C1 and C2. There were genotype x challenge x time interactions (P < 0.05) for BT, glucose and TNFα. During C1, BT increased earlier and peaked higher in RA than UH or CH, glucose increased less in RA than UH or CH, and TNFα increased more in CH than UH and RA. Glucose, TNFα, and BT did not differ (P > 0.10) among genotypes during C2. There was an interaction of genotype and challenge for IL-6 as response in UH was greater than in CH or RA in C1 but there was no effect of genotype on IL-6 in C2. There was a trend for NOX (P = 0.10) to be less in RA than in UH or CH and a trend (P = 0.10) for a genotype × challenge interaction for XO as XO was greater in UH than in CH and RA in C1 but not in C2. Results indicate the reduced response during a repeated challenge decreases the ability to detect an effect of genotype on LPS. Regardless, results indicate genotype affects bovine response to LPS and that the effect of genotype differs among the response variables assessed in this study.