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This study investigated the effect of caffeine ingestion on antigen‐stimulated T‐ (CD4⁺ and CD8⁺) and natural killer (NK)‐ (CD3⁻CD56⁺) cell activation after prolonged, strenuous cycling. In a randomized cross‐over design, nine male endurance cyclists (age: 22 ± 3 years, V̇O_2peak: 62 ± 4 mL/kg/min, mean ± SD) cycled for 90 min at 70% V̇O_2peak 60 min after ingesting 6 mg/kg body mass of caffeine (CAF) or placebo (PLA). Venous blood samples were obtained before supplementation, pre‐exercise, immediately post‐exercise and 1 h post‐exercise. Whole blood was stimulated with Pediacel (five in one) vaccine. At 1 h post‐exercise the number of antigen‐stimulated CD4⁺ cells expressing CD69 decreased on CAF compared with PLA [15 (17) × 10⁶ vs 23 (22) × 10⁶ cells/L, P<0.05]. In addition, the geometric mean fluorescence intensity (GMFI) of CD69 expression on antigen‐stimulated CD8⁺ cells …