The objective of this investigation was to evaluate the effect of magnesium (Mg) supplements in rats during prolonged restriction of motor activity (hypokinesia [HK]) and in the presence of Mg deficiency, which is characterized by increased rather than decreased plasma Mg concentration, as occurs in ambulatory conditions. The studies were performed during 98 days of HK on 100 13-week-old Sprague-Dawlay male rats weighing 360 to 390 g. They were equally divided into four groups: (1) unsupplemented control animals (UCA), (2) unsupplemented hypokinetic animals (UHA), (3) supplemented control animals (SCA), and (4) supplemented hypokinetic animals (SHA). For the simulation of the hypokinetic effect, SHA and UHA were kept for 98 days in small individual wood cages that restricted their movements in all directions without hindering food and water intake. The SCA and SHA took daily with their food an additional 0.35 mg of Mg. Before and during the hypokinetic period of 98 days, Mg in plasma, urine, and feces, balance of Mg, food intake of Mg, and body weight were determined at different intervals. In SHA and UHA, plasma Mg concentration and excretion of Mg in urine and feces increased significantly compared with SCA and UCA. Magnesium balance was negative in UHA and AHA throughout the hypokinetic period. Body weight and a food intake decreased significantly in SHA and UHA when compared with SCA and UCA. Significant losses of Mg in SHA and UHA occurred in the presence of Mg deficiency and suggest that prolonged HK induces another factor that influences Mg metabolism. We conclude that prolonged HK causes significant changes in Mg values of plasma, urine, and feces and a negative Mg balance in rats, despite Mg supplements leading to Mg deficiency.