In order to find markers to assess the functional state of the cardiovascular system before and after spaceflight (first and seventh day after landing), we analyzed the urine proteome in ten cosmonauts aged of 35 to 51 years who have completed 169 to 199-day spaceflight onboard the ISS. A special sample preparation was performed, followed by liquid chromatography-mass spectrometry. Liquid chromatography-mass spectrometry of the minor proteins was performed on a nano-HPLC Agilent 1100 system (Agilent Technologies Inc., USA) in combination with a LTQ-FT Ultra mass spectrometer (Thermo, Germany). A total of 238 proteins was identified. According to the TIGER database, a tissue origin was established for 129 proteins. We identified 20 proteins related to the cardiovascular system. It was found that changes in cosmonauts' urine proteome comprehensively reflect the adaptive responses of cardiovascular, renal and neuroendocrine systems to long-term microgravity conditions.