Many of the common diseases such as diabetes and hypertension have an effect on the microcirculation and further on the rheological properties of blood flow. Deformable red blood cells (RBCs), suspended in plasma, make up of the total blood volume. Depending on various parameters such as diameter of the vessel, hematocrit, and flow rate, blood can form a plasma layer free of its cells near the wall. The cell-free layer (CFL) which consists mainly of plasma leads to a decreased apparent viscosity of blood. This effect is widely known as the Fåhraeus–Lindqvist effect. It is one of the major factors that contribute to the fluid resistance in the microcirculation and has been extensively investigated, especially in glass microchannels.1,2 The CFL thickness is in the range of a few microns making it challenging to measure. It is commonly measured by light microscopy employing high-speed cameras. Furthermore, to provide better sensitivity, especially in confocal microscopes, RBCs are often labeled with fluorescent markers.3,4 Labeling increases the complexity of a measurement preparation process and can have an influence on the flow properties. Recently, advanced computer algorithms were developed for analyzing images and defining the CFL thickness.5 In the case of high hematocrit levels, the microscopy images are highly distorted and have low contrast. RBCs located out of the focus plain can cause shading. Thus, it is difficult to accurately determine a width between a lumen boundary and an outermost edge of a concentrated RBC suspension in the core. Currently, high-speed optical coherence tomography (OCT) system has achieved axial resolutions of to visualize tissue at cellular level.6 An extremely high resolution enables new applications in the blood rheology including study of CFL and blood flow properties at the level of a single cell. Potential applications in blood monitoring also include measuring hemoglobin7 and glucose8 concentrations. The microflows have been extensively studied in complex vessels by Doppler OCT.9–13 The spatial distribution of RBCs in microchannels has been previously evaluated with OCT.14,15 Due to an insufficient axial resolution of the used OCT systems, the thickness of a CFL has not been measured previously.