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, V̇O2peak: 62 ± 4 mL/kg/min, mean ± SD) cycled for 90 min at 70% V̇O2peak 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 decreased on CAF compared with PLA 1 h post-exercise [78 (10)% vs 102 (24)%, P<0.05]. At the same time-point GMFI of CD69 expression on antigen-stimulated CD3−CD56+ cells was increased on CAF compared with PLA [103 (9)% vs 87 (8)%, P<0.05]. These findings suggest that caffeine reduces antigen-stimulated CD69 expression on T cells while at the same time increases NK-cell activation 1 h after intensive cycling.