Pilot Study Observes Significant Biomarker Changes in Triathletes After Beetroot-Based Supplementation

A pilot study published in the journal Nutrients observed significant changes in several biomarkers related to nitric oxide and redox activity in amateur male triathletes following one week of supplementation with a beetroot-based product. The supplement was reported to be well-tolerated. The study did not measure endurance performance, stamina, or recovery outcomes.

The researchers concluded that larger, placebo-controlled trials are needed to determine if the observed biochemical changes translate to functional benefits for athletes.

Study Design and Participants

The research was a randomized, open-label, crossover trial conducted by Italian researchers.

• Participants: Ten healthy, amateur male triathletes aged 30–59 participated. All trained for a minimum of 300 minutes per week and maintained stable diets and activity levels throughout the study.
• Protocol: The study consisted of two seven-day phases: a supplement phase and a control phase. These were separated by a 15-day interval. Participants were randomized into two sequences, with each serving as their own control.
• Supplement: The daily supplement contained beetroot-derived nitrate, L-citrulline, L-arginine, carbohydrate, and N-acetylcysteine. The dose was adjusted by participant body weight: 10 grams (≤80 kg), 20 grams (80–90 kg), or 30 grams (>90 kg).
• Measurements: Blood and urine samples were collected on days 1, 7, 22, and 28 to analyze biomarkers.

Reported Biomarker Changes

After one week of supplementation, researchers reported significant increases in the following biomarkers compared to the control phase:

• Nitric oxide metabolites (NOx): 155% increase
• Peroxynitrite: 60% increase
• Inducible nitric oxide synthase (iNOS): 56% increase
• 3-nitrotyrosine (3-NT): approximately 9.0% increase
• Interleukin-6 (IL-6): 73% increase
• Reactive oxygen species (ROS): 413% increase

Acute intake was also noted to rapidly elevate iNOS and peroxynitrite levels.

Additional Observations and Tolerability

• Markers for lipid peroxidation remained stable throughout the study. The researchers reported this indicated no overt oxidative damage was detected.
• The supplement was reported to be well-tolerated. One participant reported mild loose stools, which investigators did not classify as an adverse effect. No other adverse events were recorded.

Study Limitations and Researcher Context

The study authors identified several limitations to their work:

• The sample size was small (10 participants).
• The cohort included only male participants.
• The study used an open-label design rather than a placebo-controlled one.
• The research did not measure direct endurance performance, stamina, or recovery outcomes.

In the study, the authors provide context for the observed biomarker changes. They note that in trained individuals, such transient increases in reactive oxygen species, peroxynitrite, and cytokines may reflect a coordinated redox and immune response or adaptive signaling processes rather than harmful inflammation.

Background and Suggested Future Research

Nitric oxide is involved in vascular regulation, metabolism, and skeletal muscle function. Dietary nitrate sources, such as beetroot, have been studied for their potential to increase nitric oxide availability, though existing evidence has been described as limited and heterogeneous.

The researchers suggested future studies should include longer follow-up periods, the inclusion of women and younger athletes, and the combination of molecular, physiological, and direct performance measures.