Competitive athletes regularly use a range of nutritional supplementation products in an effort to improve their sporting performance. These supplements are often used despite any knowledge on whether they actually have any effect on performance. Recently the use of beetroot juice has shown promise as an effective supplementation strategy for endurance athletes, and in particular cyclists of moderate fitness. However, there is little knowledge on the effectiveness of beetroot juice on the performance of highly trained cyclists. Therefore, the purpose of this thesis was to determine the effectiveness of nitrate supplementation on cycling and physiological responses in highly-trained cyclists. A series of studies were undertaken during which the performance and physiological responses of highly-trained cyclists were assessed before, and after a range of beetroot juice supplementation strategies. Collectively, the studies in this thesis reveal that the effectiveness of NO3-supplementation on physiological and performance measures appears to be sensitive to the training status of athletes, and the environmental conditions that the event is conducted in. In particular, despite previous positive findings in cyclists of lesser aerobic ability, any potential improvement in sporting performance or enhanced physiological responses appear to diminish in cyclists of greater aerobic ability. As such, the findings of this thesis suggest that effects of NO3- supplementation appear not to influence performance or a range of physiological measures to the same extent as lesser-trained populations.