Lower limbs show acute fluid shift in response to transition from upright to supine body position. It is hypothesized that this would affect tomographic estimations of muscle mass and composition. Seven healthy subjects were investigated during the initial 120 min of bed rest, using repeated computerized tomography (CT) and continuous bioelectrical impedance analysis (BIA). Thigh and calf muscle cross-sectional area (CSA) decreased (P < 0.05) by 1.9 and 5.5% whereas fat CSA decreased (P < 0.05) by 4.1 and 4.4%, respectively. Radiological density (RD) of muscle showed a simultaneous increase (P < 0.05) by 4.8% in calf but not (P > 0.05) in thigh. No changes occurred (P > 0.05) in muscle or fat CSA or muscle RD in either thigh or calf between the first and second hour of bed rest. Fluid shift, as estimated by BIA, showed an exponential decay in thigh (τth = 30 min) and calf (τc2 = 37 min) by 2.5 and 8.7%, respectively, from first to 120 min of bed rest. Moreover, the calf showed an initial rapid (τc1 = 8 s) 2.2% decrease. The demonstrated short-term changes in leg CSA were more pronounced in the calf than in the thigh. They were similar in muscle and subcutaneous fat. These fluid shifts merit consideration when tomographic imaging techniques are used to estimate muscle mass and composition.