Physical stress induced in healthy volunteers by the Loughborough intermittent shuttle test (LIST) was used to validate a newly developed whole-blood cell culture system (Instant leukocyte culture system (ILCS). Exercise induced immune modulation was investigated through measurement of cytokine levels after activating leukocytes in peripheral blood ex vivo using the physiologic stimulant lipopolysaccharide (LPS). LPS induced production of three different cytokines, interferon gamma (IFNgamma), interleukin-6 (IL-6), and interleukin-10 (IL-10). IFNgamma levels were significantly decreased (P = 0.02 and P = 0.001 ) and IL-10 levels significantly increased (P= 0.04 and 0.03) after exercise. LPS induced IL-6 production was only marginally further increased by exercise. In conclusion, the ILCS system provided a reliable ex vivo method, showing common as well as subject specific features in the time course of the immune modulation caused by the LIST protocol. This system will be useful for studies of the elderly, where cytokine standardisation is notoriously difficult.