Acute exercise increases red blood cell-nitric oxide synthase (RBC-NOS) activation and RBC deformability but the effect of regular training remains unclear. To detect the chronic effect of enduring moderate and high intensity training on the RBC-NOS/NO pathway and to detect a relation between RBC deformability and endurance capacity. 38 healthy male subjects were randomly assigned to one of three training groups: High Volume Training (HVT; 120-140 beats per minute (bpm)), High Intensity Training (HIT; 160-180 bpm) and Moderate Intensity Training (MIT; 140-160 bpm). Blood parameters, maximum oxygen capacity (VO2 max), RBC deformability, RBC nitrite level and RBC-NOS activation were measured after venous blood sampling at rest pre (T0) and after six weeks of training (T1). RBC-NOS activation, RBC nitrite concentration and RBC deformability were significantly increased at T1 in the HIT group. Parameters were unaltered in MIT and HVT. Maximum oxygen uptake was only significantly increased in the HIT group and regression analysis revealed positive regression between VO2 max and RBC deformability. High intensity training was the only training programme that sustainably affected RBC-NOS dependent NO production and performance capacity. HIT therefore represents a time efficient training program resulting in improved RBC function potentially improving physical condition.