Red blood cell is an important role model of microcirculation, gas exchange, drug carrier, and related diseases. There has been an increasing interest in understanding the biochemical and structural changes in Red blood cells (RBC) due to simulated microgravity conditions. In the present work, isolated human red blood cells were exposed to simulated microgravity (SMG) conditions using 2D clinostat. Comparative analysis of normal RBC and RBC in simulated microgravity conditions was performed using UV-visible spectroscopy, FTIR spectroscopy, and single-beam optical tweezer. The change in position of the Soret band absorption peak is observed after the exposure to simulated microgravity. However, biochemical functional groups of RBCs were studied using FTIR spectroscopy with a change in % Transmittance. Power spectrum density (PSD) variations in optically trapped control and SMG exposed single RBC are obtained from position signals acquired by Thorlabs’s OTKBFM-CAL data acquisition software. Microgravity (SMG)-induced stress is responsible for a reduction in haematocrit, haemolysis, change in the morphology, and rigidity of red blood cells. These investigation of the physicochemical properties of RBC in simulated microgravity is useful for improving the health of astronauts during space missions.