In the mammalian circulatory system, the viscosity of blood is a well-known determinant of vessel resistance and the frictional force (shear stress) exerted on endothelial cells lining the wall of arterial blood vessels. Shear stress is regarded as a fundamental haemodynamic signal in the modulation of blood vessel calibre, gene and protein expression of endothelial cells, and the synthesis of nitric oxide, a potent vasodilator and antiatherogenic molecule. Despite the physiological significance of shear stress in the circulatory system, it is inherently difficult to accurately quantify macrovascular shear stress in vivo due to the dynamic interaction between blood viscosity and shear rate. Indeed, blood is a non-Newtonian, shear-thinning fluid, whereby blood viscosity is reduced with increased shear rate, and vice versa, owing to the aggregation and deformability of erythrocytes. Put simply, blood viscosity cannot be characterised by a single value, and is dependent upon the instantaneous shear rate in the blood vessel. In Experiment 1, we quantified the brachial artery shear stress response across a range of metabolic demands elicited by graded rhythmic handgrip exercise. To accurately assess shear stress, measured blood viscosity data were fitted with a two-phase exponential decay, facilitating interpolation of blood viscosity values corresponding to the ultrasound-derived shear rate. The results revealed that brachial artery shear stress increased in a stepwise manner with increasing exercise workload (20%, 40%, 60% and 80% of maximum external workload). In contrast, shear rate-specific blood viscosity decreased relative to baseline (rest in normoxia) across all exercise workloads, despite concomitant haemoconcentration. Therefore, shear rate, but not blood viscosity, explained the increase in brachial artery shear stress in response to active hyperaemia. This shear-thinning behaviour of blood, consequent to increased shear stress-specific erythrocyte deformability, blunted the expected increase in shear stress based on shear rate prediction. Consequently, the use of shear stress yielded a higher slope for the brachial artery stimulus versus dilatation relationship than shear rate. Collectively, the data refute the use of shear rate to infer arterial shear stress-mediated processes. The erythrocyte, or red blood cell (RBC), is well-known for the transportation of respiratory gases within the mammalian circulatory system. Similar to endothelial cells, erythrocytes are constantly exposed to haemodynamic forces in vivo, including shear stress, when transiting the expansive vascular network. Such haemodynamic forces result in passive deformation of the flexible erythrocyte membrane, a property essential for effective convective and diffusive microvascular oxygen transport. Previous in vitro experiments have demonstrated that, in response to increased cellular deformation within the physiological range (0.1-15 Pascals [Pa]), human erythrocytes synthesise nitric oxide via activation of endothelial nitric oxide synthase (RBC-NOS). There is emerging evidence from animal models implicating a functional role of nitric oxide generated from RBC-NOS in the regulation of blood vessel calibre, independent of endothelial nitric oxide synthase (NOS). In Experiment 2, we assessed the impact of acute elevations in local shear stress and erythrocyte deformation, elicited by acute systemic hypoxaemia and brief rhythmic handgrip exercise in isolation and in combination, on the phosphorylation of RBC-NOS at serine residue 1177 (RBC-NOS1177), the primary activation site for the enzyme. Systemic hypoxaemia provides a nonmechanical stimulus that elicits a compensatory increase in arterial blood flow and shear stress. Relative to normoxic rest, normoxic handgrip exercise and systemic hypoxaemia independently amplified RBC-NOS1177 activation in tandem with increased brachial artery blood flow, shear stress and erythrocyte deformability. However, the combination of exercise and systemic hypoxaemia failed to increase RBC-NOS1177 activation beyond systemic hypoxaemia alone and normoxic exercise in the face of elevated brachial artery blood flow, shear stress and erythrocyte deformability. In fact, for the exercise performed in the systemic hypoxaemia trial, participants tended to fall into two categories: half presented with an increased phosphorylation of RBC-NOS1177, while the remaining exhibited a decrease in phosphorylation of RBC-NOS1177, when compared to normoxic exercise. The data yields novel insights into how local haemodynamic forces and oxygen availability modulate RBC-NOS1177 activation in vivo. In summary, Experiments 1 and 2 advance our understanding of the interaction between the medium (blood and erythrocytes) and peripheral haemodynamics in vivo in response to physiological stressors.