Biomarkers in motion: cytokine and flow-cytometric profiling of exercise-associated immune phenotypes in Arabian racehorses across training and racing phases

Background High-intensity exercise is associated with rapid leukocyte redistribution, oxidative responses, and transient changes in soluble mediators. Longitudinal data describing these responses across matched training sessions and official racing in Arabian racehorses remain limited. Methods Eighteen Arabian racehorses were assessed in a within-horse repeated-measures design during the first monitored high-intensity gallop of the training season (T1), a matched high-intensity gallop after 6–10 weeks of regular training (T2), and official racing (R). Blood was collected at rest and after exercise (immediately and 30–40 min after exercise). Hematological assessment included leukocyte indices and the granulocyte-to-lymphocyte ratio (GLR), whereas biochemical analyses included lactate, glucose, total protein, albumin, and globulin concentrations. Peripheral blood mononuclear cells were analyzed by flow cytometry for T-cell subsets, FoxP3-expressing and Ki-67-expressing lymphocyte populations, monocyte subpopulations, and ROS production. Serum cytokines and chemokines were measured using a multiplex equine panel. Within-horse relative fold changes were analyzed with repeated-measures approaches. Results Exercise context was associated with distinct immune and metabolic response patterns. Racing induced leukocytosis ( p = 0.0003), granulocytosis ( p = 0.0001), and delayed post-race GLR elevation ( p = 0.014). Lactate clearance was lower after racing than after T1 and T2 ( p = 0.034 for both comparisons). Glucose increased markedly after racing ( p = 8.63 × 10⁻ 7 ) while a modest decrease was observed after T1 (p = 0.045). Flow cytometry revealed exercise-context-dependent immune redistribution. CD8⁺ T cells increased after T1 ( p = 9.75 × 10⁻ 6 ) and racing ( p = 1.5 × 10⁻ 5 ), whereas CD4⁺ T cells decreased after T1 ( p = 0.0035) but increased after T2 ( p = 1.5 × 10⁻ 5 ) and racing ( p = 1.22 × 10⁻ 4 ). CD4⁺FoxP3⁺ and CD8⁺FoxP3⁺ lymphocyte populations increased after T2 ( p = 0.047 and p = 0.027, respectively). CD14⁺MHCII⁻ monocytes increased after T1 and racing, whereas CD14⁻MHCII⁺ cells increased after T2 ( p < 0.001) and decreased after T1 and racing ( p < 0.05). Racing increased the proportion of ROS-producing monocytes (p = 0.0047) and ROS intensity in lymphocytes ( p = 5.6 × 10⁻ 7 ). Quantifiable cytokine responses varied among analytes; following the race, IL-10 increased ( p = 4.8 × 10⁻ 4 ), whereas IL-8 ( p = 0.014) and GRO ( p = 0.02) decreased. Conclusions Training phase and racing were associated with distinct short-term patterns of leukocyte redistribution, stimulated ROS-generating capacity, and metabolic load. Racing produced the clearest physiological challenge, whereas T1 and T2 differed in several circulating lymphocyte and monocyte phenotypes.