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Introduction This study investigated the effects of a beet nitric oxide enhancing (NOE) supplement comprised of nitrite and nitrate on cycling performance indices in trained cyclists. Methods: Subjects completed a lactate threshold test and a high-intensity interval (HIIT) protocol at 50% above functional threshold power with or without oral NOE supplement. Results: NOE supplementation enhanced lactate threshold by 7.2% (Placebo = 191.6 ± 37.3 W, NOE = 205.3 ± 39.9; p = 0.01; Effect Size (ES) = 0.40). During the HIIT protocol, NOE supplementation improved time to exhaustion 18% (Placebo = 1251 ± 562s, NOE = 1474 ± 504s; p = 0.02; ES = 0.42) and total energy expended 22.3% (Placebo = 251 ± 48.6 kJ, NOE = 306.6 ± 55.2 kJ; p = 0.01; ES = 1.079). NOE supplementation increased the intervals completed (Placebo = 7.00 ± 2.5, NOE = 8.14 ± 2.4; p = 0.03; ES = 0.42) and distance cycled (Placebo = 10.9 ± 4.0 km, NOE = 13.5 ± 3.9 km; p = 0.01; ES = 0.65). Also, target power was achieved at a higher cadence during the HIIT work and rest periods (p = 0.02), which enhanced muscle oxygen saturation (SmO2) recovery. Time-to-fatigue was negatively correlated with the degree of SmO2, desaturation during the HIIT work interval segment (r = −0.67; p 0.008), while both SmO2 desaturation and the SmO2 starting work segment saturation level correlated with a cyclist's kJ expended (SmO2 desaturation: r = −0.51, p = 0.06; SmO2 starting saturation: r = 0.59, p = 0.03). Conclusion: NOE supplementation containing beet nitrite and nitrate enhanced submaximal (lactate threshold) and HIIT maximal effort work. The NOE supplementation resulted in a cyclist riding at higher cadence rates with lower absolute torque values at the same power during both the work and rest periods, which in-turn delayed over-all fatigue and improved total work output., Graphical abstract Image 1, Highlights • Beet nitric oxide enhancing supplementation comprised of inorganic nitrite and nitrate, but relying predominately on nitrite for nitric oxide production, improved lactate threshold power by 7.2% compared to placebo in trained cyclists and triathletes. • Beet nitric oxide enhancing supplementation increased the total number of work intervals completed while improving time to exhaustion 18%, the total energy expenditure expended during high intensity intervals 22.3%, and the total cycling distance completed 23.9% compared to placebo. • Beet nitric oxide enhancing supplementation improved performance by allowing each subject to maintain interval power targets at higher cadences and lower absolute torque which in turn enhanced SmO2 recovery during the rest intervals. • A beet nitric oxide enhancing supplement with an appropriate concentration of inorganic nitrite can be an effective ergogenic aid, and response faster than a nitrate supplement.