Hypokinesia triggers oxidative stress and accelerates the turnover of the glutathione system via the γ-glutamyl cycle. Our study aimed to identify the regulatory checkpoints controlling intracellular glutathione levels. We measured the intermediate substrates of the γ-glutamyl cycle in erythrocytes from 19 healthy young male volunteers before and during a 10-day experimental bed rest. Additionally, we tracked changes in glutathione levels and specific metabolite ratios up to 21 days of bed rest. Using gas chromatography-mass spectrometry and the internal standard technique, we observed a 9 ± 9% decrease in glutathione levels during the first 5 days of bed rest, followed by an 11 ± 9% increase from the 5th to the 10th day, nearly returning to baseline ambulatory levels. The cysteinyl-glycine-to-glutathione ratio, reflecting γ-glutamyl cyclotransferase activity (a key enzyme in glutathione breakdown), rose by 14 ± 22% in the first 5 days and then fell by 10 ± 14% over the subsequent 5 days, again approaching baseline levels. Additionally, the γ-glutamyl cysteine-to-cysteine ratio, indicative of γ-glutamyl cysteine synthetase activity (crucial for glutathione synthesis), increased by 12 ± 30% on day 5 and by 29 ± 41% on day 10 of bed rest. The results observed on day 21 of bed rest confirm those seen on day 10. By calculating the ratio of product concentration to precursor concentration, we assessed the efficiency of these key enzymes in glutathione turnover. These results were corroborated by directly measuring glutathione synthesis and degradation rates in vivo using stable isotope techniques. Our findings reveal significant changes in glutathione kinetics during the initial days of bed rest and identify potential therapeutic targets for maintaining glutathione levels.