The variable innate immune response between animals can be due to a variety of causes. Infections during the neonatal period could affect an animal’s innate response phenotype. Ten Holstein calves were used to investigate the sustained effects of neonatal LPS exposure on the innate immune system. At 8d of age, 5 calves received an IV infusion of LPS (0.5 μg/kg), while the remaining 5 received saline. A subsequent dose of LPS (0.25 μg/kg) was given to all 10 calves at 30 d of age to determine if there was any lasting effects from the first LPS challenge. Skin biopsies were collected from all calves at 22 d of age to isolate dermal fibroblasts for a cell model to explore genetic and/or epigenetic changes that may have resulted from the early life LPS treatment. Two hours after the first challenge, LPS-treated calves had greater (P < 0.05) plasma IL-6 (14.4 ± 2.8 vs. 0.6 ± 0.3 ng/mL) and TNF-α (3.2 ± 1.0 vs. 0.2 ± 0.1 ng/mL) compared to calves that had received saline, respectively. Response curves of these plasma cytokines from measurements at 0, 1, 2, 3, 5, and 7 h following the second LPS challenge were similar (P > 0.05) between groups. Rectal temperatures were not significantly different between the treatment groups in either challenge. Dermal fibroblasts isolated from LPS- or saline-treated calves produced similar (P > 0.05) levels of IL-6 (318.4 ± 96.8 vs. 359.3 ± 54.6 pg/mL) and IL-8 (183.6 ± 33.8 vs. 157.1 ± 11.7 pg/mL), respectively, in response to a 24h LPS challenge (500 ng/mL). Although the fibroblasts produced much greater levels of IL-6 (5178.6 ± 637.9 vs. 5353.4 ± 853.1 pg/mL) and IL-8 (1554.2 ± 96.8 vs. 1596.9 ± 93.4 pg/mL), respectively, in response to a 24h challenge with IL-1b (10 ng/mL), there was no early-life treatment effect. There was substantial inter-animal variation in clinical symptoms following the LPS challenges, but the early-life exposure to LPS at 8d of age did not clearly influence the response to a subsequent LPS challenge in 30-d-old calves, or affect the ability of fibroblasts to respond in vitro to LPS or IL-1b. More work is needed to determine how early-life exposure to infection may influence an animal’s innate immune response.