Effect of Beetroot Nitrate Supplementation on Nitric Oxide Pathways and Oxy-Inflammatory Biomarkers in Amateur Triathletes: A Randomized Cross-Over Pilot Study

Background/Objectives: Nitric oxide (NO) is a key mediator of vascular, metabolic, and redox pathways, influencing exercise performance. Beetroot, a natural source of inorganic nitrate, increases NO bioavailability and may modulate oxidative stress and inflammation, though data in endurance athletes remain limited. The aim of this study was to assess the effects of a novel beetroot-based nitrate supplement (B-bNs) on NO metabolism, oxidative stress, and inflammation in non-professional triathletes. Methods: This was a randomized 2 × 2 cross-over pilot study with two 7-day periods (B-bNs vs. No treatment), separated by a 15-day washout (4 visits: Day 1, 7, 22 and 28). Samples were collected at baseline (T0), 2 h post-first dose (T1), and after 7 days (T2) for the supplementation period (B-bNs) and at T0 and T2 for the “no treatment” period. The following biomarkers from plasma and urine were evaluated: NO pathway (NO metabolites (NOx), nitrite (NO2), inducible nitric oxide synthase (iNOS), peroxynitrite, 3-nitrotyrosine (3-NT)), oxidative stress (reactive oxygen species (ROS) production, 8-isoprostane, superoxide dismutase (SOD) activity), and cytokines (IL-6, IL-10). A total of 10 male triathletes (mean age 48.1 ± 9.8 years and BMI 23.9 ± 2.2 kg/m2) participated in this study. Results: No adverse events were reported. After 7 days of supplementation (T2 vs. T0), significant increases in NOx in plasma and urine (about +155%), iNOS (+56%), peroxynitrite (+60%), 3-NT (+8.6%), ROS (+413%) and IL-6 (+73%) were recorded. These values resulted significantly higher compared to “no treatment” (all p = 0.002), with no significant differences for 3-NT, SOD, 8-isoprostane, IL-6, and IL-10. Conclusions: Beetroot-based nitrate supplementation may enhance the NO-related pathway in non-professional endurance athletes with nitric-peroxydation activation, occurring without evidence of lipid oxidative damage. Larger placebo-controlled trials with standardized diet/training and performance outcomes are needed to determine the functional significance of these preliminary findings. This study was registered in the ISRCTN registry (ISRCTN10885376).