Blood flow produces fluid shear stress (SS), a frictional force parallel to the blood flow, on the endothelial cell (EC) layer of the lumen of the vessels. ECs themselves are sensitive to this SS in terms of directionality and intensity. The aim of this study was to determine the physiological SS value during the cardiac cycle and EC polarity and orientation from blood flow in healthy male and female mouse carotid artery. All procedures were done according to current national and European legislation, and agreed upon by the local ethical committee. Experiments were performed on 8 male and 8 female 8-week-old C5BL/6J mice. Measurements of maximum blood velocity and vessel diameter in diastole and systole of the right common carotid artery were performed in vivo by Doppler ultrasound imaging on isoflurane-anesthetized mice. Blood samples were then collected for determination of plasmatic and total blood viscosity and hematocrit, using a newly developed device [1]. After animal euthanasia, the right common carotid was dissected for confocal imaging after labeling the EC nucleus and Golgi apparatus. Diastolic and systolic SS values were calculated from maximal velocity, vessel diameter, and viscosity values applying a newly developed method assuming heterogeneous blood flow, i.e., a red cell central plug flow surrounded by a peripheral plasma sheath flow, initially evidenced by Fahraeus [2] (F-method), compared with the classical method considering total blood as a “Newtonian” homogenous fluid with constant viscosity (N-method) [3]. F- and N-methods differed in blood velocity profile (including hematocrit in the F-method) and viscosity values, plasmatic and total, respectively. EC orientation was defined as the angle of the nucleus-Golgi apparatus vector to the blood flow direction, classified as dromic [0;60°], antidromic [120°;180°], and lateral [60°;120°], and compared to random distribution. EC polarity was defined by the length of this vector and nucleus elongation (long/short axis ratio). Statistical comparisons were done using Mann-Whitney and Chi² tests when appropriate and considered significant for P<0.05. Total blood and plasmatic viscosity were 4±0.5 cP and 1.27 cP, respectively. Diastolic and systolic SS, calculated by F-method, were 6±2.5 Pa and 30±6.5 Pa, respectively. Diastolic and systolic SS, calculated by the N-method, were, respectively, 6% and 14% higher than by the F-method. Total blood and plasmatic viscosity were 4±0.5 cP and 1.27 cP, respectively (table 1). ECs were significantly oriented against blood flow but not polarized. No sex difference was identified, whatever the parameters. Our results showed that both methods, though based on different theoretical assumptions, were convergent in their SS calculated values. However, the F-method seems more accurate since it considers the hematocrit value whereas the N-method does not. The cardiac cycle is characterized by high rate and amplitude changes in WSS values and ECs are sensitive to the direction of the blood flow, without sex difference.