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AIM In this work, we aim to assess red blood cells (RBCs) interaction, using an approach with interchanging suspending media. METHODS Optical tweezers (OT) coupled with a microfluidic chamber allow the optically trapped RBCs to be moved between two different solutions. A cell doublet was formed in one of the solutions and then moved to another solution. The corresponding resultant change in the interaction forces was quantified. Different combinations of macromolecule solutions (plasma, dextran solution, fibrinogen, and/or albumin solution) and PBS were studied. RESULTS The doublet formed in a macromolecule solution disaggregated immediately as soon as it was moved to PBS. However in the opposite way, when cells were moved from PBS to a macromolecule solution, the cells started to interact immediately. Moving the cells from a solution with lower macromolecule concentration to a solution with higher concentration resulted in an enhanced interaction of the cells. When the cells were moved in the opposite way their interaction almost did not decrease. CONCLUSION The study reveals that red blood cells interaction can be further assessed in well-defined and interchanging media using OT coupled with a microfluidic chamber. The underlying cell-cell interaction mechanisms will be discussed.