Purpose: To determine the effect of dietary nitrate (NO₃⁻) supplementation on physiology and performance in well-trained cyclists following six to eight-days of NO₃⁻ supplementation. Methods: Eight competitive male cyclists (mean ± SD; age = 26 ± 8 y; body mass = 76.7 ± 6.9 kg; VO2peak = 63 ± 4 ml.kg⁻¹.min⁻¹) participated in a double-blind, placebo-controlled, crossover-design study in which participants ingested 70 ml beetroot juice containing ~4 mmol NO₃⁻ (NIT) or a NO₃⁻ depleted placebo (PLA) , each for 8-days. Replicating pre-treatment measures, participants undertook an incremental ramp assessment to determine VO₂peak, first (VT₁), and second (VT₂) ventilatory thresholds on day 6 (NIT6 and PLA6), moderate-intensity cycling economy on day 7 (NIT7 and PLA7), and a 4-km time-trial on day 8 (NIT8 and PLA8). Results: Relative to PLA, 6 days of NIT supplementation produced unclear effects for VO2peak (mean ±95%CL: 1.8 ±5.5%) and VT1 (3.7 ±12.3%) and trivial effects for both VT2 (-1.0 ±3.0%) and exercise economy on day 7 (-1.0 ±1.6%). However, effects for time-trial performance time (0.7 ±0.9%), and power (2.4 ±2.5%), on day 8 were likely beneficial. Conclusions: Despite mostly unclear outcomes for standard physiological determinants of performance, 8-days of NO₃⁻ supplementation resulted in likely beneficial improvements to 4-km time-trial performance in well trained male endurance cyclists.