The effect of hypokinesia on lipid metabolism in adipose tissue

The increase of nonesterified fatty acid (NEFA) concentration in plasma was observed in rats subjected to hypokinesia for 1-60 days. In the period of recovery (7 and 21 days after 60 days immobilization) the content of NEFA returned to control values. The increase of fatty acid release from adipose tissue was observed in hypokinetic rats, however the stimulation of lipolysis by norepinephrine was lower in rats exposed to hypokinesis. The decrease of the binding capacity and a diminished number of beta-adrenergic receptors were found in animals after hypokinesia. The augmentation of the incorporation of glucose into lipids and the marked increase in the stimulation of lipogenesis by insulin were found in adipose tissue of rats subjected to long-term hypokinesia. These results showed an important effect of hypokinesia on lipid mobilization, on lipogenesis and on the processes of hormone regulation in adipose tissue.     

Mineralization of human bone tissue under hypokinesia and physical exercise with calcium supplements

It has been suggested that physical exercise and calcium supplements may be used to prevent demineralization of bone tissue under hypokinesia (diminished muscular activity). Thus, the aim of this study was to determine mineral content of bones of 12 physically healthy men aged 19-24 years under 90 days of hypokinesia and intensive physical exercise (PE) with calcium lactate (C) supplements. They were divided into experimental and control groups with 6 men in each. The experimental group of men were subjected to hypokinesia (HK) and intensive PE and took 650 mg C 6 times per day; the control group was placed under pure HK, i.e. without the use of any preventive measures. The mineral content of different bone tissues was measured with a densitometric X-ray method in milligrams of calcium per 1 mm3 before and after exposure to HK. The level of bone density of the examined bone tissues decreased by 7-9% and 5-7% for the control and experimental groups of men, respectively. A statistical analysis revealed that the reduction of bone mineralization was significant with P < 0.01 in both groups of men. A comparison between bone density changes in the control and experimental groups of men failed to demonstrate significant differences. It was concluded that the level of mineralization of bone tissues decreased under hypokinesia and physical exercise with calcium supplements.     

Effect of fluid and salt supplements in preventing the development of "osteopenia" in hypokinetic rats

It has been suggested that a daily intake of fluid and salt supplements may be used to prevent bone demineralization in human subjects after prolonged exposure to hypokinesia (diminished muscular activity). Thus, the objective of this investigation was to evaluate the effect of fluid and salt supplementation in the prevention of development of osteoporosis in 64 Wistar rats with an initial body weight of 339-345 g, after exposure to 90 days of hypokinesia. They divided into 4 equal groups: the first group of rats placed under ordinary vivarium conditions and served as vivarium control; the second group were also placed under ordinary vivarium conditions but received daily fluid and salt supplements; the third group were subjected to pure hypokinesia, i.e. without the use of any preventive measures; and the fourth group were submitted to hypokinesia and received daily fluid and salt supplements. For the simulation of the hypokinetic effect the experimental group of rats were kept in small, individual, wooden cages. Through the experimental period the second and fourth group of rats received 8 ml/100 g body wt water and 5 ml 100 g body wt NaCl daily. By the end of the experimental period the animals were decapitated and the spongy matter of tibia and vertebrae of the rats were examined for changes referable to osteoporosis. It was found that the daily intake of fluid and salt supplements caused an increase in the volume density of primary spongiosa of bones. It was concluded that a daily intake of fluid and salt supplements may be used to prevent the development of osteoporosis in rats subjected to prolonged motor activity restriction.     

Electrolytes in femur and plasma of rats during prolonged restriction of motor activity.

Hypokinesia (decreased motor activity) induces insignificant bone mineral changes. The aim of this study was to measure mineralization, density, and also electrolyte content in the femur of rats during prolonged hypokinesia (HK). Studies were done on 144 male Wistar rats (370-390 g) during 15 days period of pre-HK and 90 days period of HK. Rats were equally divided into two groups: hypokinetic rats (HKR) and vivarium control (VCR). The HKR group of rats was kept in small individual cages. Femur mineralization density, ash mineral content, calcium (Ca) and phosphate (P) content, and plasma Ca and P concentration were measured. In the HKR group body weight, femur mineralization, density, ash mineral content, Ca and P concentration decreased significantly (p < or = 0.01) while plasma Ca and P concentration increased significantly (p < or = 0.01) when compared with the VCR group. The measured parameters did not change significantly in the VCR group when compared with the baseline control values. It was concluded that prolonged HK induces a significant reduction in electrolyte concentration accompanied by decreased mineralization, density, and ash mineral content of the femur of rats.     

Effect of potassium and calcium loading on healthy subjects under hypokinesia and physical exercise with fluid and salt supplements

The objective of this investigation was to determine the acute responses to the electrolyte challenges under hypokinesia and physical exercise (PE) of different intensities with fluid and salt supplementation (FSS). The studies were performed on 12 physically healthy male volunteers aged 19-24 years under 364 days of hypokinesia (decreased number of steps per day) with a set of PE with FSS. The volunteers were divided into two equal groups. The first group was subjected to a set of intensive PE and the second group was submitted to a set of moderate PE. Both groups of subjects consumed daily water and salt supplements that aimed to increase the body hydration level. For simulation of the hypokinetic effect all subjects were kept under an average of 3000 steps per day. Functional tests with a potassium chloride (KCl) and calcium lactate (Cal) load were performed during the hypokinetic period of 364 days and the 60-day, prehypokinetic period that served as control, while both groups of subjects consumed daily calcium and potassium supplements. The concentration of electrolyte and hormone levels in the blood and their excretion rate in urine were determined. Renal excretion of calcium and potassium and the blood concentration thereof increased markedly in both groups of subjects. With the potassium chloride load tests the increased potassium excretion was accompanied by higher aldosterone and insulin blood levels, and with the calcium lactate load tests the increased calcium excretion was accompanied by a decreased parathyroid content in the blood. FSS and PE, regardless of intensity, failed to attenuate calcium and potassium losses. Additional intake of KCl and Cal also failed to normalize potassium and calcium abnormalities. It was concluded that during the KCl and Cal loading tests, the increased losses of potassium and calcium in the hypokinetic subjects were due to the inability of their bodies to retain these electrolytes, and that electrolyte abnormalities could not be reversed by PE or rehydration in individuals subjected to prolonged restriction of motor activity.     

Fluid electrolyte excretion during different hypokinetic body positions of trained subjects

The aim of this study was to evaluate the effect of different body positions on renal excretion of fluid and electrolytes after exposure to 364 days of decreased number of steps per day (hypokinesia, HK). The studies were performed on 18 endurance trained male volunteers aged 19-24 years who had an average of VO2max 67 ml/kg body/min. All volunteers were divided into three equal groups: the 1st group subjected to 12 h orthostatic position (OP) and 12 h clinostatic position (CP)/day, the 2nd group exposed to 8 h orthostatic position and 14 h clinostatic position/day, and the 3rd group submitted to 10 h orthostatic position and 16 h clinostatic position/day for 364 days. For the simulation of the hypokinetic effect all volunteers were kept under an average of 3000 steps/day for 364 days. Diuresis and the concentrations of sodium, potassium, chloride, calcium and magnesium as well as excretion of creatine were determined in 24-h urine samples. By the end of the hypokinetic period all volunteers, regardless of their body position during HK, manifested a significant increase in renal excretion of fluid and electrolytes as compared to prehypokinetic period values. It was concluded that prolonged restriction of motor activity induced a significant increase in renal excretion of fluid and electrolytes in endurance trained subjects regardless to their body position and duration thereof per day.     

Fluid-electrolyte changes in physically conditioned subjects after hypokinesia and chronic hyperhydration

The aim of this study was to determine whether fluid-electrolyte changes, which are developed during prolonged hypokinesia (decreased number of km per day), can be prevented or minimized with the use of a daily intake of fluid and salt supplementation (FSS). The experiments on hypokinesia (HK) were performed for 364 days on 18 endurance-trained male volunteers in the age range of 21-23 years, with an average maximum oxygen uptake of 67 ml kg-1. All volunteers were divided into three equal groups: six volunteers were placed on a continuous regime of exercise of 14.0 km day-1 and served as control subjects. Six volunteers were subjected to continuous HK without FSS and were considered as the unsupplemented hypokinetic subjects (UHS). The remaining volunteers were under continuous HK and FSS and were considered as the supplemented hypokinetic subjects (SHS). For the simulation of the hypokinetic effect, the UHS and SHS groups were kept continuously under an average of 2.7 km day-1 for the duration of the experiment. Prior to exposure to HK, all volunteers were on the same exercise regime as the controls. During the pre-experimental period of 60 days and during the post-experimental period, urinary excretion of electrolytes and concentrations of sodium, potassium, calcium and magnesium in serum as well as serum osmolality were determined. An increased renal excretion of fluid and electrolytes and a decreased serum electrolyte concentration were observed in the SHS, while a decreased renal excretion of fluid and electrolytes and an increased serum electrolyte concentration were observed in the UHS, during the initial stages of the post-hypokinetic period. By day 30 of the post-hypokinetic period these changes were reverted back to the control levels. We concluded that chronic hyperhydration may be used to attenuate urinary and serum electrolyte changes in endurance-trained volunteers after exposure to prolonged HK.     

Effect of hyperhydration on bone mineralization in physically healthy subjects after prolonged restriction of motor activity

The objective of this investigation was to evaluate the effect of a daily intake of fluid and salt supplementation (FSS) on bone mineralization in physically healthy male volunteers after exposure to hypokinesia (decreased number of steps taken/day) over a period of 364 days. The studies were performed after exposure to 364 days of hypokinesia (HK) on 18 physically healthy male volunteers who had an average VO2max of 65 ml/kg/min and were aged between 19 and 24 years. For the simulation of the hypokinetic effect the volunteers were kept under an average of 1000 steps/day. The subjects were divided into three equal groups of 6: 6 underwent a normal ambulatory life (control group), 6 were placed under HK (hypokinetic group) and the remaining 6 were subjected to HK and consumed a daily FSS (water 26 ml/kg body wt and NaCl 0.10 mg/kg body wt) (hyperhydrated group). The density of the ulnar, radius, tibia, fibular, lumbar vertebrae and calcenous was measured. Calcium and phosphorus changes, plasma volume, blood pressure and body weight were determined. Calcium content in the examined skeletal bones decreased more in the hypokinetic subjects than in the hyperhydrated subjects. Urinary calcium and phosphorus losses were more pronounced in hypokinetic than hyperhydrated subjects. Plasma volume and body weight increased in hyperhydrated subjects, while it decreased in hypokinetic subjects. It was concluded that a daily intake of FSS may be used to neutralize bone demineralization in physically healthy subjects during prolonged restriction of motor activity.     

Gender differences in organ density in a rat simulated microgravity model

Research investigating the physiological effects of microgravity on the human body has demonstrated a shift of body fluids in actual spaceflight and in simulated Earth-based microgravity models in both males and females, possibly causing many deleterious physiological effects. Twenty-five anatomically normal female (NF) and 20 ovariectomized (OE) Fischer 344 rats were randomly selected to be in an experimental (1 h of 45 degrees head-down tilt, 45HDT) or control (1 h of prone position) group. At the end of the hour experimental period, the density of the brain, lungs, heart, liver, and left and right kidneys were measured using spiral computed tomography (SCT) while the rats remained in their experimental positions. A sub-group of OE rats (N=6) was administered estrogen replacement therapy on a daily basis (5 micrograms/kg body weight, s.c.) for 4 days and then underwent 1 h of 45HDT and SCT analysis at one day, 2 days, and 5 days to determine if estrogen replacement therapy would alter organ densities. Our data demonstrate that 1 h of 45HDT produced significant increases (p<0.05) in the organ densities of the brain, liver, left kidney, and lung of the OE female group compared to their prone controls. However, only the brain density was significantly increased in the NF group. Estrogen replacement therapy caused a significant decrease in brain organ density at the 5 day time point compared to the 24 h time point. We conclude that estrogen plays a role in fluid distribution in a rat 45HDT model.     

Chronic hyperhydration and hematological changes in trained subjects during prolonged restriction of motor activity

The objective of this investigation was to evaluate the effect of a daily intake of fluid and salt supplementation (FSS) on the hemoglobin content of endurance trained athletes during hypokinesia (decreased number of steps from 10,000 to 3000 steps per day). The studies were performed on 30 long-distance runners who had a VO2max average of 66 ml kg-1 min-1 and were in the age range of 19-24 years. Prior to their exposure to hypokinesia (HK) of 364 days, all volunteers were on an average of 10,000 steps per day. All volunteers were divided into three equal groups: the first group underwent normal ambulatory life (control subjects), the second group was kept under continuous restriction of motor activity (hypokinetic subjects), and the third group was placed under continuous restriction of motor activity and consumed 26 ml water kg-1 body weight daily and 0.1 g sodium chloride kg-1 body weight in the form of supplementation (hyperhydrated subjects). For simulation of the hypokinetic effect, the number of steps taken per day by the second and third groups of volunteers was restricted to an average of 3000. During the hypokinetic period we determined reticulocytes (Rt), hemoglobin (Hb), hematocrit (Hct), plasma volume (PV), red blood cell (RBC) mass and VO2max. In hyperhydrated volunteers the content of Hb and Hct decreased significantly, while PV, RBC mass and Rt count increased significantly. In hypokinetic volunteers Hb and Hct increased, while PV, RBC and Rt decreased significantly. It was concluded that chronic hyperhydration may be used to attentuate an increase in the Hb content of physically conditioned subjects during prolonged restriction of motor activity.     

Renal excretion of water in men under hypokinesia and physical exercise with fluid and salt supplementation

It has been suggested that under hypokinesia (reduced number of steps/day) and intensive physical exercise, the intensification of fluid excretion in men is apparently caused as a result of the inability of the body to retain optimum amounts of water. Thus, to evaluate this hypothesis, studies were performed with the use of fluid and sodium chloride (NaCl) supplements on 12 highly trained physically healthy male volunteers aged 19-24 years under 364 days of hypokinesis (HK) and a set of intensive physical exercises (PE). They were divided into two groups with 6 volunteers per group. The first group of subjects were submitted to HK and took daily fluid and salt supplements in very small doses and the second group of volunteers were subjected to intensive PE and fluid-salt supplements. For the simulation of the hypokinetic effect, both groups of subjects were kept under an average of 4000 steps/day. During the prehypokinetic period of 60 days and under the hypokinetic period of 364 days water consumed and eliminated in urine by the men, water content in blood, plasma volume, rate of glomerular filtration, renal blood flow, osmotic concentration of urine and blood were measured. Under HK, the rate of renal excretion of water increased considerably in both groups. The additional fluid and salt intake failed to normalize water balance adequately under HK and PE. It was concluded that negative water balance evidently resulted not from shortage of water in the diet but from the inability of the body to retain optimum amounts of fluid under HK and a set of intensive PEs.     

ESA's Biopan 1--"Vitamin" experiment preliminary results

The purpose of “Vitamin” experiment is to study the efficiency of protective substances on three biological acellular systems aqueous solutions exposed to cosmic radiation in space. The first system “LDL”is a low density lipoprotein. The second is “E2-TeBG complexe” in which estradiol (E2) is bound to its plasmatic carrier protein, testosterone-estradiol binding globulin (TeBG). The third is “pBR 322”, a plasmid. “Vitamin” experiment was accomodated in the Biopan which had been mounted on the outer surface of a Foton retrievable satellite. The experiment was exposed to space environment during 15 days. A stable temperature of about 20 °C was maintained throughout the flight. “Vitamin” experiment preliminary results are presented and discussed.     

Fluid electrolyte and hormonal changes in conditioned and unconditioned men under hypokinesia

It has been suggested that hypokinesia (diminished muscular activity) may induce more changes in fluid electrolyte metabolism and hormonal concentration of blood plasma in conditioned than unconditioned men. Thus, the objective of this investigation was to determine the effect of 7 days of hypokinesis (HK) on fluid-electrolyte excretion and hormonal content of blood in 12 physically healthy men aged 19-23 years. They were divided into two equal groups according to their physical conditioning. For the simulation of the hypokinetic effect the men were kept under a rigorous bed rest regime. During the background period (BGP), that is prior to the exposure to HK, and under HK, the rate of elimination of fluid, sodium and potassium, and the content of blood plasma aldosterone and cortisol was measured. The amount of excretion of fluid and electrolytes increased while blood plasma aldosterone content decreased. In the conditioned men, a greater excretion of fluid and electrolytes and a greater reduction of plasma aldosterone concentration was observed. It was concluded that hypokinesia induced substantial changes in fluid-electrolyte excretion and hormonal content of blood plasma in both conditioned and unconditioned men.     

The effects of hypokinesia in primates on bone strength.

A 14 days ground based primate hypokinesia investigation was conducted to determine what changes, if any, in bone strength may be demonstrated following 2 weeks cast immobilization and at 14 davs after removal from casts. Controlled compressive loads were applied to vertebral bodies excised from control, 2 weeks immobilization and 2 weeks post immobilization Rhesus monkeys. The material properties reported herein are ultimate load, displacement to ultimate load, stiffness and energy to ultimate load. These results show a decrease in bone strength following immobilization and a still further decrease in bone strength after 14 days.     

Thyrotropin receptor and membrane interactions in FRTL-5 thyroid cell strain in microgravity

The aim of this work was to analyze the possible alteration of thyrotropin (TSH) receptors in microgravity, which could explain the absence of thyroid cell proliferation in the space environment. Several forms of the TSH receptor are localized on the plasma membrane associated with caveolae and lipid rafts. The TSH regulates the fluidity of the cell membrane and the presence of its receptors in microdomains that are rich in sphingomyelin and cholesterol. TSH also stimulates cyclic adenosine monophosphate (cAMP) accumulation and cell proliferation. Reported here are the results of an experiment in which the FRTL-5 thyroid cell line was exposed to microgravity during the Texus-44 mission (launched February 7, 2008, from Kiruna, Sweden). When the parabolic flight brought the sounding rocket to an altitude of 264?km, the culture media were injected with or without TSH in the different samples, and weightlessness prevailed on board for 6 minutes and 19 seconds. Control experiments were performed, in parallel, in an onboard 1g centrifuge and on the ground in Kiruna laboratory. Cell morphology and function were analyzed. Results show that in microgravity conditions the cells do not respond to TSH treatment and present an irregular shape with condensed chromatin, a modification of the cell membrane with shedding of the TSH receptor in the culture medium, and an increase of sphingomyelin-synthase and Bax proteins. It is possible that real microgravity induces a rearrangement of specific sections of the cell membrane, which act as platforms for molecular receptors, thus influencing thyroid cell function in astronauts during space missions.     

Choroidal responses in microgravity. (SLS-1, SLS-2 and hindlimb-suspension experiments)

Fluid and electrolyte shifts occuring during human spaceflight have been reported and investigated at the level of blood, cardio-vascular and renal responses. Very few data were available concerning the cerebral fluid and electrolyte adaptation to microgravity, even in animal models. It is the reason why we developed several studies focused on the effects of spaceflight (SLS-1 and SLS-2 programs, carried on NASA STS 40 and 56 missions, which were 9- and 14-day flights, respectively), on structural and functional features of choroid plexuses, organs which secrete 70–90 % of cerebrospinal fluid (CSF) and which are involved in brain homeostasis. Rats flown aboard space shuttles were sacrificed either in space (SLS-2 experiment, on flight day 13) or 4–8 hours after landing (SLS-1 and SLS-2 experiments). Quantitative autoradiography performed by microdensitometry and image analysis, showed that lateral and third ventricle choroid plexuses from rats flown for SLS-1 experiment demonstrated an increased number (about x 2) of binding sites to natriuretic peptides (which are known to be involved in mechanisms regulating CSF production). Using electron microscopy and immunocytochemistry, we studied the cellular response of choroid plexuses, which produce cerebrospinal fluid (CSF) in brain lateral, third and fourth ventricles. We demonstrated that spaceflight (SLS-2 experiment, inflight samples) induces changes in the choroidal cell structure (apical microvilli, kinocilia organization, vesicle accumulation) and protein distribution or expression (carbonic anhydrase II, water channels,…). These observations suggested a loss of choroidal cell polarity and a decrease in CSF secretion. Hindlimb-suspended rats displayed similar choroidal changes. All together, these results support the hypothesis of a modified CSF production in rats during long-term (9, 13 or 14 days) adaptations to microgravity.     

Severe disruption of the cytoskeleton and immunologically relevant surface molecules in a human macrophageal cell line in microgravity—Results of an in vitro experiment on board of the Shenzhou-8 space mission

During spaceflight the immune system is one of the most affected systems of the human body. During the SIMBOX (Science in Microgravity Box) mission on Shenzhou-8, we investigated microgravity-associated long-term alterations in macrophageal cells, the most important effector cells of the immune system. We analyzed the effect of long-term microgravity on the cytoskeleton and immunologically relevant surface molecules. Human U937 cells were differentiated into a macrophageal phenotype and exposed to microgravity or 1g on a reference centrifuge on-orbit for 5 days. After on-orbit fixation, the samples were analyzed with immunocytochemical staining and confocal microscopy after landing. The unmanned Shenzhou-8 spacecraft was launched on board a Long March 2F (CZ-2F) rocket from the Jiuquan Satellite Launch Center (JSLC) and landed after a 17-day-mission. We found a severely disturbed actin cytoskeleton, disorganized tubulin and distinctly reduced expression of CD18, CD36 and MHC-II after the 5 days in microgravity. The disturbed cytoskeleton, the loss of surface receptors for bacteria recognition, the activation of T lymphocytes, the loss of an important scavenger receptor and of antigen-presenting molecules could represent a dysfunctional macrophage phenotype. This phenotype in microgravity would be not capable of migrating or recognizing and attacking pathogens, and it would no longer activate the specific immune system, which could be investigated in functional assays. Obviously, the results have to be interpreted with caution as the model system has some limitations and due to numerous technical and biological restrictions (e.g. 23 °C and no CO₂ supply during in-flight incubation). All parameter were carefully pre-tested on ground. Therefore, the experiment could be adapted to the experimental conditions available on Shenzhou-8.     

Effect of weightlessness on sympathetic-adrenomedullary activity of rats.

Three cosmic experiments were performed in which rats spent 18-20 days in space on board the biosatellites "COSMOS 782", "COSMOS 936" and "COSMOS 1129". The following indicators of the sympathetic-adrenomedullary system (SAS) activity were measured: tissue and plasma catecholamines (CA), CA-synthesizing enzymes--tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenylethanolamine-N-methyltransferase (PNMT)--as well as CA-degrading enzymes-monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). Adrenal epinephrine (EPI) and norepinephrine (NE) as well as CA-synthesizing and degrading enzymes were not significantly changed in the animals after flight on COSMOS 782. On the other hand, a significant increase was found in heart CA, the indicator which is usually decreased after stress. 26 days after landing all values were at control levels. The results obtained, compared to our previous stress experiments on Earth, suggest that prolonged weightlessness does not appear to be a pronounced stressful stimulus for the SAS. Heart and plasma CA, mainly NE, were increased both in the group living in the state of weightlessness and the group living in a centrifuge and exposed to artificial gravitation 1 g (COSMOS 936), suggesting again that prolonged weightlessness is not an intensive stressful stimulus for the SAS. The animals exposed after space flight on COSMOS 1129 to repeated immobilization stress on Earth showed a significant decrease of adrenal EPI and an expressive increase of adrenal TH activity compared to stressed animals which were not in space. Thus, the results corroborate that prolonged state of weightlessness during space flight though not representing by itself an intensive stressful stimulus for the sympathetic-adrenomedullary system, was found to potentiate the response of "cosmic rats" to stress exposure after return to Earth.     

Effects of muscle electrostimulation during simulated weightlessness

In a 45-day experiment test subjects were exposed to bed rest with their heads down at -4 degrees C. Twice a day their muscles of the stomach, back, femur, and shin were stimulated with electric current for 25-30 min. The value of muscle tension was close to their maximum voluntary contraction. The main objective was to prevent muscle atrophy and to maintain their trophic and functional state. Physiological measurements were carried out together with morphological, cytochemical, and biometric evaluations. The tissue removed during biopsy from M. soleus 7 days before the test and on the 30th hypokinetic day was used as substrate. Electrostimulation affected favourably the tone and strength of muscles as well as their static and dynamic endurance. Morphological studies showed a positive effect of electrostimulation on the muscle tissue, preventing the development of atrophic processes. During the first post-hypokinetic day orthostatic tolerance increased.     

Immobilization induces a very rapid increase in osteoclast activity

We studied in a randomized, strictly controlled cross-over design, the effects of 6 days 6 degrees head-down tilt bed rest (HDT) in eight male healthy subjects in our metabolic ward. The study consisted of two periods (phases) of 11 days each in order to allow for the test subjects being their own controls. Both study phases were identical with respect to environmental conditions, study protocol and diet. Two days before arriving in the metabolic ward the subjects started with a diet. The diet was continued in the metabolic ward. The metabolic ward period (1l days) was divided into three parts: 4 ambulatory days, 6 days either HDT or control and 1 recovery day. Continuous urine collection started on the first day in the metabolic ward to analyze calcium excretion and bone resorption markers. On the 2nd ambulatory day in the metabolic ward and on the 5th day in HDT or control blood was drawn to analyze serum calcium, parathyroid hormone, and bone formation markers. Urinary calcium excretion was, as early as the first day in immobilization, increased (p<0.01). CTX- and NTX-excretion stayed unchanged in the first 24 h in HDT compared to the control. But already on the 2nd day of immobilization, both bone resorption markers significantly increased. We conclude from these results--pronounced rise of bone resorption markers--that already 24 h of immobilization induce a significant rise in osteoclast activity in healthy subjects. Thus, it appears possible to use short-term bed rest studies as a first step for the development of countermeasures to immobilization.