Cows on concrete flooring (vs. rubber) have classical immunological indicators of chronic pain; increased PBMC numbers, more IL-1β, and lower IL-1Ra. We hypothesized that a profile of biomarkers on lymphocytes and monocytes could be associated with chronic pain. 1st-calf heifers were assigned to rubber or concrete flooring. On day 45 and 90 of the 1st lactation, hematology and lymphocyte marker expression were analyzed. Differential counts were similar to our previous study, numbers and percentages of lymphocyte and monocytes were greater for cows on concrete than rubber. However, PMN % decreased on day 90 for cows on concrete. Eosinophil % and numbers were greater on both days for cows on rubber and basophils increased for cows on rubber at day 90. Buffy coat cells were further stimulated with substance-P (SP) in vitro. Cells were labeled with CD4, CD8, CD3, CD14, WC-1 (ɣδ), and DEC-205 (dendritic cells, DC). Many 3-way interactions were detected for gated lymphocytes (P < 0.05). CD4, CD8, and DC intensity was most affected by the flooring, day, and SP stimulation. Additionally, CD14 of gated lymphocytes was greater for cows on rubber and also decreased with SP stimulation, suggesting that chronic pain decreases CD14 expression. Although antigen presentation appears to be most affected by DC changes, CD4 and CD8 intensity differed by floor type and SP stimulation. ɣδ T-cells intensity was not influenced by the flooring, time, or SP. A differential response by floor type and day was detected, but overall, lymphocytes from cows on concrete were more sensitive to SP. In gated monocyte populations, DC, CD4, and CD8 intensity increased with time. Rubber flooring also increased DC intensity. SP had differential effects on DC and CD4 intensity by flooring. This chronic pain model showed that chronic pain predominantly affects antigen presenting cells and CD4 and CD8 lymphocytes.