Mass spectrometry–based proteomics was employed to analyze urine from eight healthy volunteers during a 21-day bed rest (BR) study. The analysis included trypsinolysis in solution prior to liquid chromatography and tandem mass spectrometry (LC-MS/MS), and spectrum processing using the bioinformatic tools. Relying on 221 IPI indices with scores from 24 to 1700, 169 different proteins were identified. Molecular functions, biological processes, and cell components as the loci of certain protein functioning were determined with the help of UniProt-GOA. Associative interactions networks were constructed using BiNGO. There were 14 proteins identified that were functional in the cardiovascular system mostly. They were annotated, and the dynamics of their occurrence throughout the experiment was considered. Grounding on the biological functions of these proteins and an assumption of eligible activation of different biological processes during BR was made.