The circulating number of natural killer (NK) cells largely changes after an acute bout of physical exercise. Granulysin is a cytolytic granule protein with a broad range of antimicrobial and tumoricidal activities produced and released by human NK cells and cytolytic T lymphocytes. Since NK cells constitutively produce granulysin, most serum granulysin in healthy humans is derived from NK cells. Serum graulysin levels in the healthy humans may therefore reflect the size of whole-body NK cell population in the body. The aim of this study was to determine the effect of an acute bout of exhaustive exercise on serum granulysin in comparison with the circulating number of NK cells. Six healthy, young male volunteers participated in the study. Each subject underwent both exhaustive exercise and resting sessions in a random order with at least a seven-day interval. Subjects were asked to run to exhaustion on a treadmill with an incremental graded protocol. Blood samples were collected before, immediately after, and 1 hr, 3 hr, 6 hr, 12 hr and 24 hr after exercise. Serum granulysin levels were measured by enzyme-linked immunosorbent assay (ELISA). NK cells were determined by flow cytometry. Exhaustive exercise induced a 4.8-fold increase in peripheral blood NK cells, but no significant change in serum granulysin. Our results support the hypothesis that exhaustive exercise-induced changes in the circulating number of NK cells represent a redistribution of lymphocytes, rather than the change in the size of whole-body NK cell population.