Carboxyfluorescein diacetate succinamidyl ester (CFSE) labelling of lymphocyte populations can provide unique insights into cell function at rest and with exercise due to its ability to quantify cell division on an individual cell basis. This study aimed to characterise the effect of long duration intense exercise on T-lymphocyte function. Well-trained endurance runners completed 60 minutes of treadmill running at 95% of individual anaerobic threshold. Blood samples were collected: before exercise, after 30 and 60 minutes of exercise and after 30, 60 and 90 minutes of recovery. Isolated peripheral blood mononuclear cells were labelled with CFSE and cultured with or without mitogen (PHA). Following culture, cell suspensions were labelled with CD3 (APC), CD8 (PE) and expansion rates and cell death rates were calculated for each sample as well as mitosis rates for each cell generation. Exercise was associated with a 60% decrease in cell expansion in both CD4 and CD8 cell types from before exercise to mid-exercise (p<0.05). The significant decrease in expansion rate in the mid-exercise samples for both cell types was mirrored by a 65% increase in cell death (p<0.05) in both cell types at that sample point. Exercise had no effect on the mitosis rate of either CD4 or CD8 cells in any cell generation (G0-G3). This study indicates that one-hour of intense exercise affects in vitro T-lymphocyte function. These data suggest for the first time that exercise decreases cell expansion rate via an increase in cell death of both CD4 and CD8 T-lymphocytes, rather than a decrease in mitosis.