To study the effects of exercise on circulating leukocytes and leukocyte subsets, physically active (n = 32) and sedentary (n = 32) male and female subjects were randomly assigned to an exercise or control condition. Exercise involved a continuous incremental protocol consisting of cycling for three periods of 6 min at power outputs corresponding to 55%, 70% and 85% maximal oxygen uptake (VO2max). Blood samples were drawn from a venous catheter at baseline, and at 6 min, 12 min, and 18 min after beginning the exercise and 2 h following completion of exercise. Resting- and exercise-induced alterations in total leukocytes were independent of gender and subject fitness level. Relative to baseline, each increment in workload resulted in a rapid increase in the number of circulating leukocytes. Increases in neutrophils, lymphocytes and monocytes accounted for the exercise-induced leukocytosis. With regard to lymphocytes, exercise resulted in a significant increase in the number of T cells (CD3+), T helper cells (CD4+), T suppresser (CD8+) and natural killer (NK) cells (CD3-/CD16+/CD56+). The largest percentage increase occurred in the NK cell population. The CD4+: CD8+ ratio decreased (P < 0.001) throughout exercise due to a larger increase in the number of CD8+ cells relative to CD4+ cells. An exercise-induced neutrophilia, lymphocytopenia, and eosinophelia was observed 2 h into recovery. Exercise resulted in significant increases in plasma epinephrine and norepinephrine levels. There was no indication of a hypothalamic-pituitarty-adrenal response during exercise. The results indicate that the rapid, albeit transient, alteration in the number of circulating leukocytes during and following an acute progressive incremental exercise test are independent of gender and fitness.