Highlights: • The effects of a nutritional protein restriction (NPR) in prepubertal female lambs are studied. • The NPR didn't affect the animals' first lactation production traits nor the SCC response to a mammary inflammatory challenge. • The NPR showed a significant influence on eight of the 14 plasma biomarkers analysed in the inflammatory challenge. • The NPR animals showed a slower inflammatory systemic response and a larger functional alteration of the mammary gland. • These results could be explained by the VEGF-A biomarker, involved in vasculogenesis during mammary gland development. Abstract: This study evaluated the influence of a temporary nutritional protein restriction (NPR) performed, under commercial conditions, in prepubertal female lambs on first lactation milk production traits and the inflammatory response triggered by an inflammatory challenge of the. From 40 Assaf female lambs, we defined a control group (C n = 20), which received a standard diet for replacement lambs and the NPR group (n = 20), which received the same diet but without soybean meal between 3 and 5 months of age. About 150 days after lambing, 24 of these ewes (13 NPR, 11C) were subjected to an intramammary infusion of E. coli lipopolysaccharide (LPS). Our dynamic study identified indicator traits of local (SCC) and systemic (rectal T a , IL-6, CXCL8, IL-10, IL-36RA, VEGF-A) response to the LPS challenge. The NPR did not show significant effects on milk production traits and did not affect the SCC and rectal T a after the LPS challenge. However, the NPR had a significant influence on 8 of the 14 plasma biomarkers analysed, in all the cases with higher relative values in the C group. The effects observed on VEGF-A (involved in vasculogenesis during mammary gland development and vascular permeability) and IL-10 (a regulatory cytokine classically known by its anti-inflammatory action) are the most remarkable to explain the differences found between groups. Whereas further studies should be undertaken to confirm these results, our findings are of interest considering the current concern about the future world's demand for protein and the need for animal production systems to evolve toward sustainability.