Magnesium (Mg) is the second most abundant intracellular cation with modulating properties in a number of metabolic processes, e.g. in glycolysis, and intracellular signalling processes, e.g. regulation of ion channels and transporters. There are conflicting data available about the regulation of Mg in blood cells during exercise. Moreover, there are no data available about changes of the metabolic important fraction of ionized Mg(2+) both in blood and in blood cells during exercise. The present study investigated the changes of ionized Mg(2+) and total Mg concentration in different compartments after a stepwise treadmill ergometer test. Intracellular ionized Mg(2+) of thrombocytes and erythrocytes was determined by the magnesium sensitive fluorescent dyes mag-fura-2 and Mag-Green using fluorescence spectroscopy and flow cytometry, respectively. Ionized Mg(2+) in blood/serum was measured by an ion-sensitive microelectrode. Total cellular and serum Mg concentration were investigated using atomic absorbance spectroscopy and photometry, respectively. The present results shown that at the end of the ergometer test, ionized Mg(2+) in both blood and serum and total serum Mg decreased. In contrast, intracellular concentration of ionized Mg increased in both thrombocytes and erythrocytes. Total intracellular Mg was unchanged making a Mg(2+) shift between the intra- and extracellular compartment unlikely. The present study therefore demonstrated opposite changes of the ratio [ionized Mg(2+)]/[total Mg] in the intracellular and the extracellular compartment after anaerobic exercise. In in vitro experiments, similar changes of ionized Mg(2+) in both compartments could be mimicked by application of weak acids like propionic and lactic acid. It is concluded changes in the fraction of ionized Mg(2+) should be high enough to influence intracellular signalling and metabolic processes.