Does nitrate supplementation worsen endurance capacity in women? Original paper

In this randomized crossover trial involving young women, nitrate supplementation worsened endurance capacity by approximately 10%. This effect was observed during both the early and late follicular phases of the menstrual cycle.

The effect of nitrate supplementation on exercise performance in women during different phases of the menstrual cycle (i.e., the early and late follicular phases).

10 young women (average age of 24).

Participants had regular menstrual periods and were considered non-aerobically trained (VO₂peak <45 mL/kg/min). None of the participants were taking hormonal contraception.

In this randomized controlled crossover trial, the participants consumed 70 mL per day of either nitrate-rich (about 13 millimolar per day of nitrate) or nitrate-depleted beetroot juice for 3 days, followed by a bout of exercise testing 2 hours after consuming their assigned juice. This process was performed during both the early and late follicular phases of the participants’ menstrual cycle. One month later, participants repeated the process with the other juice.

Exercise testing measured the participants’ endurance capacity and exercise economy. Exercise economy was based on oxygen uptake during a 4-minute bout of cycling at moderate intensity. Endurance capacity was measured by having the participants cycle at a severe intensity (intensity was individualized based on previous testing) until exhaustion (i.e., when cycling speed dropped by more than 10 rotations per minute).

Prior to exercise testing, the investigators examined the participants’ blood pressure and pulse wave velocity (PWV, a marker of arterial stiffness), as well as plasma levels of N-oxides (e.g., nitrate and nitrite), estradiol (a major form of estrogen), and progesterone.

On average, fatigue occurred 49 seconds sooner following nitrate supplementation (430 seconds vs. 479 seconds), representing a 10% worsening in endurance capacity.

Effect of nitrate on endurance performance

EF: Early follicular phase, LF: Late follicular phase. * indicates a statistically significant difference between nitrate and placebo group

Nitrate supplementation did not affect exercise economy, blood pressure, or PWV, but it did increase plasma levels of both nitrate and nitrite.

All of the previous findings were similar during both phases of the follicular cycle. However, nitrate worsened exercise performance more during the late follicular phase than the early follicular phase (71 seconds worse vs. 28 seconds worse), but this finding was not statistically significant.

Estradiol levels were higher during the late phase compared to the early phase of the follicular cycle (298 vs. 188 picomoles per liter).

In recent years, there has been an explosion of interest in the use of nitrate-containing foods (particularly beetroot juice) as ergogenics to improve exercise performance. This was borne out of an understanding of what’s known as the nitrate-nitrite-nitric oxide pathway, the mechanism by which consuming nitrate can increase tissue levels of nitric oxide.

Upon ingestion, nitrate accumulates in the mouth both passively and via enterosalivary circulation.^[2] Oral bacteria can then metabolize nitrate into nitrite, which, when swallowed, enters the stomach and is converted into nitrogen oxides by stomach acid. These nitrogen oxides then travel into the bloodstream (likely bound to stable molecules like glutathione^[3]), where they can break down to form nitric oxide.

How nitric oxide is formed

Nitric oxide has a variety of biological effects that can theoretically improve exercise performance. In particular, nitric oxide can promote greater blood flow to muscles, potentially increasing the delivery of oxygen and nutrients critical for energy generation during exercise.^[4] Nitric oxide may also improve exercise performance by stimulating mitochondria in a manner that improves the efficiency of cellular energy generation.

Despite these supposed benefits, the current study found that nitrate worsened endurance capacity. This is more surprising when considering that in many previously conducted studies, nitrate seemed neutral or even beneficial for exercise performance. In fact, a meta-analysis of randomized controlled trials from 2021 found that nitrate supplementation improved time to exhaustion during endurance activities like cycling, with the participants able to exercise for an average of 25 seconds longer until fatigue.^[5] What could explain this discrepancy between the current study and the collective scientific literature?

For one thing, the current study involved young women. This is important, because past studies looking at the effect of nitrate on exercise performance involved mostly men. Women are highly underrepresented in nitrate research.^[6] When looking specifically at clinical trials involving women, nitrate supplementation usually does not improve markers of exercise performance.^{[7][8][9][10][1]} In fact, a randomized controlled trial from 2023 found that although nitrate supplementation increased endurance capacity by 15% in men, it had no clear effect in women.^[11]

As previously mentioned, a key factor in exercise performance is the ability to generate energy in the form of adenosine triphosphate (ATP), a process that requires oxygen. It’s been proposed that, beyond simply improving oxygen delivery to muscles, nitrate can enhance the efficiency by which mitochondria makes energy using oxygen.^[12] In line with this, nitrate supplementation sometimes reduces how much oxygen people utilize during a set amount of exercise.^[5] Conversely, the current study found that oxygen use during exercise (exercise economy) was unaffected by nitrate supplementation, suggesting that nitrate didn’t improve mitochondrial efficiency.

Interestingly, nitrate also didn’t affect the reported difficulty of exercise (rating of perceived exertion) after both 3 minutes and 5 minutes of cycling at high intensity. This could mean that, rather than nitrate promoting physical fatigue, nitrate increased cognitive fatigue, making it more difficult to keep cycling as participants approached the same level of physical difficulty.

Beyond just the muscles and the cardiovascular system, exercise performance is influenced by mental state. Greater cognitive fatigue has been shown to worsen physical endurance, often hastening time to exhaustion, despite not always increasing people’s perceived exertion. (For more about this phenomenon, check out the Editor’s Pick, “Does mental fatigue impair exercise performance?”). It’s also possible that nitrate did in fact increase perceived exertion, but the effect took longer than 5 minutes to occur.

One possible mediator of the apparent sex-specific effects of nitrate could be estrogen.^[13] Women, especially premenopausal women, typically have much higher estrogen levels than men, and estrogen has a complicated and likely important effect on nitric-oxide-generating pathways. Some researchers have even proposed that menstruating women may see a blunted or lack of response to nitrate during the late follicular phase of the menstrual cycle because high estrogen levels seem to increase nitric oxide via the L-arginine pathway, potentially preventing the metabolism of nitrate to nitric oxide.^[13] In partial agreement with this, the current study found nitrate seemed to worsen endurance capacity more during the late follicular phase (when estradiol levels were about 60% higher), although this finding was not statistically significant, perhaps due to the small sample size. Further research is therefore needed to determine whether estrogen actually influences the effects of nitrate supplementation.

Ultimately, the current study doesn’t overturn the large body of evidence suggesting nitrate can improve endurance exercise performance in some contexts. The two findings may not even be in conflict, since both can be explained by nitrate’s ergogenic effects differing between men and women. If anything, the current study should be a reminder that findings from aggregated evidence don’t always apply to everyone. Anyone (but especially women) interested in using nitrate to improve their exercise performance should consider that their experience could be disappointing and proceed accordingly.

The nitrate-rich beetroot juice contained an estimated 13 mmol (650 mg) of nitrate. This is generally considered a high enough dose of nitrate to elicit ergogenic effects.^[1]