Caffeine is consumed by many athletes for its known ergogenic properties. However despite the high intention by athletes to use caffeine (Chester & Wojek, 2008), little research has concentrated on its effects on human immune cell function following high intensity exercise. A study by Bishop et al. (2005) demonstrated that caffeine ingestion 60 min before a 90 min cycle at 70% VO_2peak increased the natural state of activation of CD4⁺ and CD8⁺ cells in vivo before and after exercise. However, this does not necessarily suggest enhanced effector function when faced with an antigenic challenge. Therefore, the aim of the present study was to investigate the effect of caffeine ingestion on human T cell (CD4⁺ and CD8⁺) function following prolonged, high intensity cycling in response to antigen stimulation, as assessed by the early activation molecule CD69. Following University Ethical Committee approval, 8 healthy male endurance trained cyclists (mean ± SD.; age 22 ± 2 years, VO_2peak 62.9 ± 3.2 ml.kg^-1.min^-1) volunteered to participate in the study. Subjects performed two exercise trials in a single-blind randomised crossover design. Each trial was performed following an overnight fast and 60 h abstention from caffeine containing foods and beverages. Each trial comprised 90 min cycling on a stationary ergometer at 70% VO_2peak 60 min after ingesting 6 mg.kg^-1 body mass of either caffeine (CAF) or dextrose placebo (PLA) in capsule form. During the trials, subjects consumed 2 ml.kg^-1 body mass of water at 15 min intervals and a further 5 ml.kg^-1 body mass 5 min post-exercise. Venous blood samples were obtained before supplementation, pre-exercise, immediately post-exercise and 1 h post-exercise. The trials were performed at least 7 days apart. Human T cells were stimulated with the Pediacel (5 in 1) vaccine at a dose of 1:4000 and 1:8000 (optimum and sub-optimum, respectively) and incubated for 20 h at 37 °C, 5% CO₂. Subsequent expression of CD4, CD8 and CD69 was analysed by flow cytometry. Results were expressed relative to the pre-supplement value and analysed using a two-factor (time x trial) repeated measures ANOVA. Post hoc t tests with Holm-Bonferroni adjustment were applied where appropriate. Statistical significance was accepted at P<0.05. Caffeine ingestion did not affect 1 h post-exercise concentrations of total lymphocytes or percentage of CD4⁺ and CD8⁺ cells. However, at 1 h post-exercise geometric mean fluorescence intensity (density of expression) for CD69 on stimulated CD4⁺ and CD8⁺ cells was significantly decreased on CAF compared with PLA (CD4⁺; PLA, 91.97% ± 20.23%; CAF, 65.94% ± 20.70%; CD8⁺: PLA, 99.77% ± 24.13%; CAF, 79.65% ± 11.85%, P<0.05, n=8). These findings suggest that caffeine ingestion before exercise inhibited antigen-stimulated CD69 expression on CD4⁺ and CD8⁺ cells 1 h after high intensity prolonged cycling.