Abstract
Background
Few studies have evaluated the association between dietary nitrate (NO₃⁻) intake and muscle strength, and none has examined this relationship using urinary nitrate as a biomarker of nitrate intake.
Aim
To investigate the association between urinary NO₃⁻ levels (a marker of NO₃⁻ consumption) and muscle strength in younger and middle-aged adults and older adults.
Methods
This cross-sectional study utilized data from the National Health and Nutrition Examination Survey (NHANES 2011–2012). A total of 1,301 individuals were included and stratified by age (< 60 years, n = 902; ≥60 years, n = 399). NO₃⁻ levels (ng/mL) in the urine were measured using ion chromatography coupled with tandem mass spectrometry and electrospray ionization. Muscle strength was assessed using combined handgrip strength (HGS), calculated as the sum of measurements from both hands. Linear regression analyses were performed to examine the association between urinary NO₃⁻ levels and HGS, adjusting for potential confounding variables.
Results
A positive association between urinary NO₃⁻ levels and HGS was observed in older adults (β = 0.00004; 95% CI: 0.00001–0.00007; p = 0.009). When urinary nitrate was normalized to urine osmolality, a positive association with HGS was also observed in older adults (β = 0.017; 95% CI: 0.0004–0.034; p = 0.046). No significant associations were found in the overall sample or among younger and middle-aged adults.
Conclusion
Urinary NO₃⁻ levels were positively associated with handgrip strength in older adults, but not in younger and middle-aged adults. Given the small magnitude of this association, longitudinal studies are warranted to determine its clinical relevance.
Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1186/s12986-026-01146-9.
Keywords: Nitric oxide, Muscle contraction, Sarcopenia, Muscle mass
Introduction
Aging is a key factor contributing to the decline in muscle strength, which is associated with an increased risk of falls, reduced quality of life, greater physical dependency, higher hospitalization rates, and increased mortality [1–5]. Multiple factors can contribute to this decline, including a pro-inflammatory state, increased oxidative stress, a sedentary lifestyle, reduced Ca²⁺ sensitivity during muscle contraction, and inadequate dietary intake [6–10]. Therefore, investigating nutritional factors that may be associated with muscle strength is of particular importance. Several dietary components have been associated with muscle strength, including protein intake, vitamin D, fat intake, and certain antioxidants [11–17]. However, the association between dietary nitrate (NO₃⁻) and muscle strength in representative population samples remains unclear. NO₃⁻ is available in the diet, primarily in vegetables such as beetroot, spinach, arugula, and radish [18]. Dietary NO₃⁻ is absorbed into the bloodstream, with ~ 25% being taken up by the salivary glands and concentrated in the saliva [19]. Anaerobic bacteria naturally residing in the crypts of the tongue convert NO₃⁻ into nitrite [20, 21]. Part of the nitrite that is swallowed undergoes reduction to nitric oxide in the acidic environment of the stomach, while a significant portion enters the bloodstream, leading to an increase in circulating nitrite levels [22, 23]. After a single dose of NO₃⁻ intake, plasma NO₃⁻ levels typically reach their peak within 1–2 h, followed by a peak in plasma nitrite concentration at around 2–3 h. Both levels then progressively decrease, returning to baseline within approximately 24 h [24]. The majority of absorbed inorganic NO₃⁻ (approximately 65%) is excreted in the urine [25, 26].
The effects of NO₃⁻ supplementation, primarily through beetroot juice, have been widely investigated, especially for its potential to lower blood pressure [27] and improve exercise performance, primarily in aerobic exercises such as cycling and running [28, 29]. Additionally, recent meta-analyses have suggested a potential effect of NO₃⁻ supplementation on muscle power and strength [30, 31], indicating that acute or chronic dietary NO₃⁻ supplementation may increase maximal muscle power in humans by an average of approximately 5%, a magnitude likely to be of considerable clinical relevance [31]. However, these studies examined the effects of acute or chronic NO₃⁻ supplementation, primarily through beetroot juice, and do not allow for extrapolation to whether individuals with higher habitual dietary NO₃⁻ intake have greater muscle strength.
Only a few studies [32–34] have investigated the association between habitual dietary NO₃⁻ intake and muscle strength or function. Hayes et al. [32] reported that habitual NO₃⁻ intake, specifically from plant-based sources, but not from animal-derived foods, was inversely associated with frailty in community-dwelling older women. Similarly, Sim et al. [33] found that higher NO₃⁻ intake was associated with lower odds of weak grip strength and slow Timed Up and Go performance in older Australian women living in the community. Longitudinal data assessing habitual dietary NO₃⁻ intake over a 12-year period also suggest that higher intake, predominantly from vegetables, may contribute to improved lower-limb muscle strength and physical function in both men and women (baseline mean age ≈ 49 years) [34]. Although these studies were well conducted, they assessed dietary intake through questionnaires, specifically food frequency questionnaires, which are known to have limitations, including imprecise quantification of food intake and inaccurate self-reporting by participants [35]. These limitations may affect the reliability of the observed associations between dietary NO₃⁻ intake and muscle strength. Therefore, studies examining this association using objective biomarkers are needed. Given that approximately 65% of dietary NO₃⁻ is excreted in the urine [25, 26], urinary NO₃⁻ may be considered a marker of NO₃⁻ intake. Furthermore, all previous studies [32–34] were conducted in community-dwelling individuals, and their findings cannot be generalized to population-based studies using representative samples. Moreover, these studies did not formally assess whether associations differ across age groups, such as younger and middle-aged adults versus older adults. This is an important gap, as older adults are more susceptible to strength decline due to age-related physiological changes, including reduced Ca²⁺ sensitivity during muscle contraction [8]. Given evidence that NO₃⁻ may influence muscle Ca²⁺ handling by enhancing calcium uptake and facilitating contraction [36, 37], it is plausible that the association between NO₃⁻ and muscle strength varies by age, being more evident in older adults than in younger and middle-aged adults, who generally have better-preserved muscle function. Therefore, the aim of the present study was to investigate the association between urinary NO₃⁻ levels and muscle strength in younger and middle-aged adults and older adults in a nationally representative sample of U.S. adults. We hypothesized that urinary NO₃⁻ levels would be positively associated with muscle strength in older adults, but not in younger and middle-aged adults.
Methods
Study design and population
This cross-sectional study utilized data from the National Health and Nutrition Examination Survey (NHANES), specifically from the 2011–2012 cycle. NHANES is an ongoing research program that collects information on the health and nutritional status of the U.S.A population through structured interviews, standardized physical examinations, and laboratory assessments. For the present study, we included individuals aged 20 to 80 years from the urinary NO₃⁻ subsample (n = 2144) who had complete data on muscle strength, anthropometric measurements, biochemical tests, dietary intake, and sociodemographic characteristics. Exclusion criteria were individuals under 20 years of age, pregnant or breastfeeding individuals, and those with missing data on urinary NO₃⁻, muscle strength, dietary intake, anthropometric measurements (weight and height), or uric acid levels. After applying these criteria (Fig. 1), a total of 1301 individuals were included and stratified by age group (< 60 years, n = 902; ≥60 years, n = 399). The cutoff of 60 years was selected because clinically meaningful declines in muscle strength are common beyond this age [38], while maintaining an adequate sample size to support multivariable adjustment. The NHANES data collection was approved by the National Center for Health Statistics Research Ethics Review Board (NCHS ERB) under protocol number 2011–17, and informed consent was obtained from all participants.
Fig. 1.
Flowchart of subjects evaluated: sample selection from NHANES 2011–2012
Urinary NO₃⁻
Urine samples were collected in polystyrene cryovials or polypropylene centrifuge tubes, with a minimum volume of 0.5 mL. Samples were transported at approximately 4 ± 3 °C and subsequently stored at − 20 ± 5 °C until analysis. Urinary NO₃⁻ levels (ng/mL) were quantified using ion chromatography coupled with tandem mass spectrometry and electrospray ionization. In a subanalysis performed in the present study, urine osmolality was also measured to calculate the urinary nitrate-to-osmolality ratio. Osmolality was determined by freezing point depression osmometry, in which a urine sample is supercooled in the osmometer’s chamber, crystallization is induced, and the release of heat of fusion raises the temperature to a stable equilibrium plateau. This plateau corresponds to the freezing point of the sample and is converted into osmolality values based on the principle that a 1.0 osmole/kg solution decreases the freezing point of water by 1.858 °C, expressed in mOsm/kg H₂O.
Muscle strength
Handgrip strength was measured using a digital handgrip dynamometer (Takei Digital Grip Strength Dynamometer, Model T.K.K. 5401). Combined handgrip strength was calculated by summing the highest value obtained from each hand. This variable was not computed for participants who performed the test with only one hand. Participants were randomly assigned to begin the test with either their dominant or non-dominant hand.
Anthropometric data
Anthropometric data were collected by trained professionals. Height was measured using a digital stadiometer, and weight was measured using a digital scale. Body mass index (BMI) was calculated as weight divided by height squared (kg/m²), following the protocol proposed by Lohman [39].
Biochemical parameters
Creatinine and serum uric acid levels were measured using a colorimetric method (Beckman UniCel® DxC800 Synchron). The estimated glomerular filtration rate (eGFR) was estimated using the equation developed by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) [40].
Dietary intake
To assess dietary intake, trained professionals administered two 24-hour dietary recalls for each participant. The first recall was conducted in person, and the second, 3 to 10 days later, by telephone. The five-step multiple-pass method [41] was used, which includes the following stages: a quick food list, a list of commonly forgotten foods, identification of times and meals, detailed descriptions and review, and a final review. Nutrient intake was calculated using the USDA Food and Nutrient Database and included total energy intake (kcal/day), carbohydrate intake (g/day), protein intake (g/day and g/kg), total fat (g/day), saturated fat (g/day), monounsaturated fat (g/day), polyunsaturated fat (g/day), total fiber (g/day), alcohol (g/day), vitamin C (mg/day), vitamin A (µg/day), vitamin K (µg/day), and vitamin E (mg/day). To account for both the in-person and telephone recalls, the average of the two was used.
Covariates of interest
Several parameters were included as confounders in the present study. The demographic covariates comprised age (years), sex (men or women), race/ethnicity (non-Hispanic White or other), educational level (college degree or not), marital status (married/living as married or single/divorced/widowed/never married), and annual household income (up to $19,999; $20,000–$54,999; $55,000–$74,999; or over $75,000). Health and behavioral factors considered were diabetes status (prediabetes, yes, or no), physical activity (categorized as yes or no), arthritis (yes or no), and smoking status (yes or no). When appropriate, a missing category was created for certain variables. Details regarding the assessment of these variables have been described previously [42]. For biochemical parameters, the eGFR was included. Adjustments for anthropometric and dietary intake data encompassed BMI (kg/m²), total energy intake (kcal/day), daily protein intake (g/day), and alcohol consumption (Had at least 12 alcohol drinks/1 year? ).
Statistical analysis
The sample was characterized according to tertiles of urinary NO₃⁻ levels and age groups (< 60 years and ≥ 60 years) (Supplementary Table 1). Continuous variables were presented as means and standard deviations, while categorical variables were expressed as percentages and confidence intervals. For categorical variables with missing values, a “missing” category was created. All analyses utilized the svy command to weight the data, accounting for the urinary NO₃⁻ sub-sample weight. Linear regression analysis was performed to evaluate the association between urinary NO₃⁻ levels (ng/ml and µg/ml) and muscle strength, both in the total sample and stratified by age. The analyses were conducted using four models: Model 1, unadjusted; Model 2, adjusted for age, sex and BMI; Model 3, which included all variables from Model 2 with additional adjustments for energy intake, protein intake, physical activity, and eGFR; and Model 4, which included all variables from Model 3 with additional adjustments for arthritis, diabetes, race/ethnicity, annual family income, marital status, educational level, smoking status, and alcohol consumption. The covariates included in the adjusted models were selected a priori on the basis of their known or plausible associations with urinary nitrate and/or handgrip strength, aiming to reduce residual confounding and strengthen the validity of the findings [43]. These analyses were also conducted in a subanalysis (total sample: n = 1266; younger and middle-aged adults: n = 874; older adults: n = 392) using the urinary nitrate-to-osmolality ratio instead of urinary nitrate alone, thereby accounting for urine concentration. A p-value < 0.05 was considered statistically significant. All analyses were conducted using Stata software version 14.0 (StataCorp, College Station, TX, USA).
Results
Characteristics of individuals according to tertiles of urinary NO₃⁻ levels
Individuals with higher urinary NO₃⁻ levels were younger, had greater body weight and height, and exhibited higher combined handgrip strength. This group also included a higher proportion of men and alcohol consumers, along with a lower proportion of menopausal women. Participants in the higher tertiles (2 or 3) showed higher eGFR and uric acid levels (mg/dL), and consumed more energy (kcal/day), protein (g/day), total fat (g/day), polyunsaturated fat (g/day), saturated fat (g/day), monounsaturated fat (g/day), alcohol (g/day), and vitamin E (mg/day) (Table 1). The characteristics of the participants according to age groups (younger and middle-aged adults, < 60 years; and older adults ≥ 60 years) are presented in Supplementary Table 1.
Table 1.
Sociodemographic, health, behavioral, anthropometric, biochemical, and dietary characteristics of the total sample and by tertiles of urinary nitrate levels
| Total sample | Tertile 1 | Tertile 2 | Tertile 3 | p-trend | |
|---|---|---|---|---|---|
| Urinary nitrate (ng/ml) | 55,595 ± 52,069 | 19,252 ± 7255 | 43,520 ± 7732 | 102,861 ± 64,098 | < 0.001 |
| Sociodemographic characteristics | |||||
| Age, y | 47.4 ± 16.9 | 49.5 ± 17.3 | 48.5 ± 17.6 | 44.2 ± 15.3 | 0.004 |
| Sex, % | 0.001 | ||||
| Women | 49.4 (46.6–52.3) | 60.7 (55.2–66.0) | 48.3 (43.1–53.5) | 42.9 (35.9–50.1) | |
| Men | 50.6 (47.7–53.4) | 39.3 (34.0–44.8) | 51.7 (46.5–56.9) | 57.1 (49.8–64.1) | |
| Race/ethnicity, % | 0.549 | ||||
| Non-hispanic white | 69.8 (61.2–77.1) | 71.2 (63.2–78.0) | 69.2 (58.7–78.0) | 69.0 (58.5–77.8) | |
| Others | 30.2 (22.9–38.8) | 28.8 (22.0–36.8) | 30.8 (22.0–41.3) | 31.0 (22.2–41.5) | |
| Educational level, % | 0.950 | ||||
| Under high school graduate | 35.0 (27.9–42.9) | 36.3 (28.7–44.7) | 32.1 (26.0–38.9) | 36.6 (25.2–49.7) | |
| Some college or over | 65.0 (57.1–72.1) | 63.6 (55.2–71.3) | 67.9 (61.1–74.0) | 63.4 (50.3–74.8) | |
| Missing | 0.01 (0.002–0.1) | 0.04 (0.005–0.4) | - | - | |
| Marital status, % | 0.959 | ||||
| Married/living with partner | 61.0 (59.6–67.2) | 59.8 (50.2–68.6) | 64.0 (57.8–69.8) | 59.6 (51.8–66.9) | |
| Single/divorced/widowed/never married | 38.9 (32.8–45.4) | 40.2 (31.4–49.8) | 36.0 (30.2–42.2) | 40.4 (33.1–48.8) | |
| Annual family income, % | 0.473 | ||||
| $0–19,999 | 17.6 (13.3–22.9) | 19.2 (14.8–24.5) | 16.4 (11.7–22.5) | 17.3 (11.8–24.6) | |
| $20,000–54,999 | 38.1 (33.5–43.0) | 35.6 (29.0–42.7) | 39.6 (32.1–47.6) | 39.2 (31.7–47.3) | |
| $55,000–74,999 | 11.3 (8.3–15.2) | 12.6 (7.5–20.4) | 9.2 (6.7–12.4) | 12.2 (7.9–18.3) | |
| Over $75,000 | 29.7 (24.8–35.1) | 29.2 (22.5–37.0) | 32.5 (26.4–39.2) | 27.6 (19.6–37.2) | |
| Missing | 3.2 (1.7–5.8) | 3.4 (1.6–7.0) | 2.4 (0.1–5.9) | 3.7 (1.7–7.9) | |
| Alcohol consumption, % | 0.005 | ||||
| No | 17.4 (14.4–21.0) | 20.5 (16.0–26.0) | 21.3 (16.5–27.0) | 10.7 (6.9–16.2) | |
| Yes | 78.2 (75.2–81.0) | 75.0 (70.8–78.9) | 75.4 (68.1–81.4) | 84.1 (79.1–88.1) | |
| Missing | 4.3 (3.0–6.2) | 4.4 (2.2–8.5) | 3.4 (1.7–6.7) | 5.1 (3.3–7.9) | |
| Health conditions and behavior | |||||
| Diabetes, % | 0.296 | ||||
| No | 87.4 (84.9–89.5) | 87.1 (82.7–90.6) | 84.4 (80.6–87.5) | 90.6 (85.6–93.9) | |
| Yes | 10.9 (9.2–12.9) | 11.7 (8.4–16.0) | 13.3 (10.9–16.2) | 7.8 (4.8–12.5) | |
| Pre-diabetes | 1.7 (0.9–3.3) | 1.1 (0.3–4.0) | 2.3 (0.9–5.9) | 1.6 (0.7–4.0) | |
| Missing | 0.02 (0.0002–0.2) | 0.06 (0.007–0.5) | - | - | |
| Hypertension, % | 0.415 | ||||
| No | 68.3 (64.0–72.4) | 65.5 (57.5–72.8) | 69.0 (67.8–75.4) | 70.4 (62.1–77.5) | |
| Yes | 31.7 (27.6–36.0) | 34.4 (27.1–42.5) | 31.0 (24.6–38.2) | 29.6 (22.5–37.9) | |
| Missing | 0.02 (0.003–0.2) | 0.07 (0.008–0.5) | - | - | |
| Menopause*, % | 0.029 | ||||
| No | 47.8 (42.4–53.2) | 39.3 (33.0–46.0) | 54.4 (46.9–62.0) | 52.2 (41.3–63.0) | |
| Yes | 44.6 (39.9–49.4) | 52.3 (42.9–61.5) | 41.1 (32.7–49.9) | 37.8 (29.9–46.4) | |
| Missing | 7.6 (5.2–11.0) | 8.4 (4.3–15.7) | 4.4 (1.9–10.0) | 9.9 (6.1–15.8) | |
| Arthritis, % | 0.307 | ||||
| No | 76.8 (72.0–81.0) | 76.1 (68.3–82.4) | 74.4 (67.3–80.3) | 79.8 (73.5–85.0) | |
| Yes | 22.9 (18.7–27.8) | 23.6 (17.2–31.4) | 25.3 (19.3–32.4) | 20.1 (15.0–26.4) | |
| Missing | 0.3 (0.2–0.3) | 0.3 (0.05–2.4) | 0.4 (0.04–0.3) | 0.1 (0.01–0.6) | |
| Physical activity, % | 0.393 | ||||
| Yes | 41.5 (37.1–46.0) | 38.9 (32.5–45.9) | 42.3 (35.1–49.9) | 43.1 (35.5–51.1) | |
| No | 58.5 (54.0–62.9) | 61.1 (54.1–67.5) | 57.7 (50.1–64.9) | 56.9 (48.9–64.5) | |
| Vigorous | 0.125 | ||||
| Yes | 19.9 (16.4–24.0) | 13.3 (8.8–19.6) | 24.2 (18.9–30.5) | 22.3 (13.9–33.7) | |
| No | 80.1 (76.0–83.6) | 86.7 (80.4–91.2) | 75.8 (69.5–81.1) | 77.7 (66.3–86.1) | |
| Moderate | 0.542 | ||||
| Yes | 36.4 (32.6–40.4) | 34.7 (28.6–41.2) | 37.1 (31.1–43.6) | 37.4 (31.1–44.1) | |
| No | 63.6 (59.6–67.4) | 65.3 (58.8–71.4) | 62.9 (56.4–68.9) | 62.6 (55.9–68.8) | |
| Smoking status, % | 0.393 | ||||
| No | 80.6 (78.0–83.0) | 82.2 (78.2–85.7) | 80.7 (74.5–85.6) | 78.9 (73.9–83.2) | |
| Yes | 19.4 (17.0–22.0) | 17.8 (14.3–21.8) | 19.3 (14.4–25.5) | 21.1 (16.8–26.1) | |
| Anthropometric characteristics | |||||
| Body mass, kg | 82.3 ± 21.7 | 78.4 ± 18.8 | 83.2 ± 22.5 | 85.3 ± 22.9 | < 0.001 |
| Height, m | 1.69 ± 0.10 | 1.67 ± 0.10 | 1.69 ± 0.10 | 1.71 ± 0.10 | < 0.001 |
| Body mass index, kg/m2 | 28.8 ± 6.7 | 28.1 ± 6.1 | 29.2 ± 7.2 | 29.1 ± 6.9 | 0.134 |
| Combined handgrip strength, kg | 72.7 ± 22.9 | 67.7 ± 20.7 | 73.3 ± 23.2 | 77.1 ± 23.6 | < 0.001 |
| Biochemical parameters | |||||
| Creatinine, mg/dL | 0.88 ± 0.30 | 0.89 ± 0.42 | 0.86 ± 0.22 | 0.88 ± 0.20 | 0.655 |
| Estimated glomerular filtration rate, ml/min/1.73 m² | 96.0 ± 21.9 | 93.1 ± 23.7 | 96.7 ± 21.5 | 98.2 ± 20.1 | 0.045 |
| Uric acid, mg/dL | 5.44 ± 1.41 | 5.30 ± 1.40 | 5.34 ± 1.43 | 5.67 ± 1.40 | 0.012 |
| Dietary intake | |||||
| Energy, kcal/d | 2139 ± 840 | 2011 ± 820 | 2213 ± 820 | 2194 ± 865 | 0.003 |
| Carbohydrate, g/d | 258.7 ± 109.8 | 248.1 ± 103.6 | 270.4 ± 110.7 | 257.9 ± 113.7 | 0.249 |
| Protein, g/d | 82.2 ± 34.3 | 77.0 ± 32.7 | 85.7 ± 34.6 | 83.9 ± 34.8 | 0.040 |
| Protein, g/kg | 1.04 ± 0.46 | 1.01 ± 0.43 | 1.07 ± 0.48 | 1.03 ± 0.46 | 0.736 |
| Total fat, g/d | 80.4 ± 37.7 | 74.3 ± 35.7 | 83.9 ± 37.8 | 82.9 ± 38.8 | 0.007 |
| Saturated fat, g/d | 25.5 ± 13.1 | 23.9 ± 12.6 | 26.8 ± 13.4 | 25.9 ± 13.1 | 0.039 |
| Monounsaturated fat, g/d | 28.8 ± 14.5 | 26.0 ± 13.1 | 30.3 ± 14.3 | 30.1 ± 15.5 | 0.001 |
| Polyunsaturated fat, g/d | 19.4 ± 10.3 | 18.3 ± 9.6 | 19.8 ± 10.5 | 20.2 ± 10.6 | 0.043 |
| Total fiber, g/d | 17.9 ± 9.7 | 17.8 ± 8.9 | 18.7 ± 10.7 | 17.3 ± 9.4 | 0.558 |
| Alcohol, g/d | 11.6 ± 25.5 | 10.1 ± 26.4 | 9.0 ± 19.3 | 15.7 ± 28.5 | 0.015 |
| Vitamin C, mg/d | 88.3 ± 89.7 | 83.0 ± 75.4 | 87.2 ± 80.9 | 94.6 ± 107.8 | 0.247 |
| Vitamin A, µg/d | 634.3 ± 442.9 | 629.8 ± 411.5 | 666.5 ± 508.9 | 607.8 ± 402.9 | 0.426 |
| Vitamin K, µg/d | 123.9 ± 135.7 | 124.7 ± 130.0 | 122.2 ± 144.7 | 124.7 ± 132.5 | 0.996 |
| Vitamin E, mg/d | 8.9 ± 5.5 | 8.2 ± 4.5 | 9.5 ± 6.4 | 9.1 ± 5.5 | 0.010 |
Data are described as mean (standard deviation) or percentage (confidence interval). Bold font indicates that the p-value is statistically significant. * Data are presented for women only
Data from NHANES 2011–2012
Association between urinary NO₃⁻ levels and muscle strength
Urinary NO₃⁻ levels (ng/ml and µg/ml) were positively associated with handgrip strength in the total sample, as well as in both younger and middle-aged adults and older adults, in Model 1 (unadjusted). However, after adjustment for basic covariates (Model 2 and Model 3) and in the fully adjusted model (Model 4), urinary NO₃⁻ levels remained positively associated with handgrip strength only in older adults, but not in the total sample or in younger and middle-aged adults (Table 2). Additional adjustment for uric acid did not alter these results (data not shown).
Table 2.
Linear regression analysis between urinary nitrate (ng/mL and µg/mL) and handgrip strength in younger and middle-aged adults and older adults
| Nitrate (ng/mL) | Model 1 | p | Model 2 | p | Model 3 | p | Model 4 | p |
|---|---|---|---|---|---|---|---|---|
| Total sample |
0.00006 (0.00003–0.0001) |
0.001 |
0.00001 (-0.00001–0.00003) |
0.343 |
0.00001 (-0.00001–0.00004) |
0.334 |
0.00001 (-0.00001–0.00004) |
0.344 |
| Younger and middle-aged adults |
0.00004 (0.000004–0.00008) |
0.033 |
0.000003 (-0.00003–0.00003) |
0.852 |
0.000004 (-0.00003–0.00004) |
0.794 |
0.000004 (-0.00003–0.00003) |
0.799 |
| Older adults |
0.0001 (0.00003–0.00016) |
0.005 |
0.00004 (0.000009–0.00007) |
0.015 |
0.00004 (0.00001–0.00007) |
0.010 |
0.00004 (0.00001–0.00007) |
0.009 |
| Nitrate (µg/mL) | ||||||||
|---|---|---|---|---|---|---|---|---|
| Total sample | 0.063 (0.029–0.097) | 0.001 | 0.011 (-0.012–0.033) | 0.343 | 0.012 (-0.013–0.036) | 0.334 | 0.011 (-0.013–0.035) | 0.344 |
| Younger and middle-aged adults | 0.039 (0.004–0.074) | 0.033 | 0.003 (-0.027–0.032) | 0.852 | 0.004 (-0.027–0.035) | 0.794 | 0.004 (-0.026–0.034) | 0.799 |
| Older adults | 0.096 (0.033–0.159) | 0.005 | 0.039 (0.009–0.070) | 0.015 | 0.041 (0.011–0.071) | 0.010 | 0.038 (0.011–0.066) | 0.009 |
Model 1: unadjusted; Model 2: age, sex and body mass index; Model 3: age, sex, body mass index, energy intake, protein intake, physical activity and eGFR; and Model 4: age, sex, body mass index, energy intake, protein intake, physical activity, eGFR, arthritis, diabetes, race/ethnicity, annual family income, marital status, educational level, smoking status and alcohol consumption. Bold font indicates that the p-value is statistically significant
Analyses using the urinary nitrate-to-osmolality ratio yielded results consistent with those obtained for urinary NO₃⁻ alone, demonstrating a positive association with muscle strength exclusively in older adults after adjustment for confounders, with larger β coefficients observed (Table 3).
Table 3.
Linear regression analysis between the urinary nitrate-to-osmolality ratio and handgrip strength in younger and middle-aged adults and older adults
| Urinary nitrate-to-osmolality ratio | Model 1 | p | Model 2 | p | Model 3 | p | Model 4 | p |
|---|---|---|---|---|---|---|---|---|
| Total Sample | 0.003 (-0.034–0.040) | 0.872 | 0.003 (-0.017–0.023) | 0.770 | 0.006 (-0.014–0.026) | 0.515 | 0.005 (-0.015–0.025) | 0.578 |
| Younger and middle-aged adults | -0.016 (-0.055–0.022) | 0.385 | -0.003 (-0.028–0.022) | 0.804 | 0.0002 (-0.024–0.024) | 0.983 | -0.0002 (-0.024–0.024) | 0.988 |
| Older Adults | 0.024 (-0.030–0.079) | 0.353 | 0.015 (-0.004–0.034) | 0.104 | 0.019 (0.001–0.037) | 0.039 | 0.017 (0.0004–0.034) | 0.046 |
Model 1: unadjusted; Model 2: age, sex and body mass index; Model 3: age, sex, body mass index, energy intake, protein intake, physical activity and eGFR; and Model 4: age, sex, body mass index, energy intake, protein intake, physical activity, eGFR, arthritis, diabetes, race/ethnicity, annual family income, marital status, educational level, smoking status and alcohol consumption. Bold font indicates that the p-value is statistically significant. Urinary nitrate-to-osmolality ratio is expressed as (ng/mL)/(mOsm/kg H₂O)
Discussion
The main finding of this study was that urinary NO₃⁻ was positively associated with muscle strength in older adults, but not in younger and middle-aged adults. This result supports our a priori hypothesis that urinary NO₃⁻ would be positively associated with muscle strength only among older adults. To our knowledge, this is the first study to evaluate the relationship between NO₃⁻ intake and muscle strength using urinary NO₃⁻ as an objective biomarker in a representative population sample.
Only a few observational studies [33, 34] have investigated the association between habitual dietary NO₃⁻ intake and muscle strength. Sim et al. [33] reported that higher NO₃⁻ intake was associated with lower odds of weak grip strength in community-dwelling older Australian women. In a subsequent study, the same research group demonstrated that habitual dietary NO₃⁻ intake was associated with improved lower-limb muscle strength over a 12-year period in both men and women, with a baseline mean age of ~ 48 years [34]. Taken together, these studies [33, 34] and the present findings support the notion that NO₃⁻ intake, regardless of the assessment method (dietary questionnaire or biomarker), is associated with muscle strength and that this relationship is consistent across different muscle strength measures (handgrip strength and lower-limb strength). It is also plausible to suggest that this association is more pronounced in older adults, given that Sim et al. [33] focused exclusively on older individuals. Moreover, although Sim et al. [34] included participants with a mean baseline age of 48.6 ± 11.1 years, the longitudinal follow-up over 12 years indicates that most participants were older adults by the end of the study period.
Consistent with this interpretation, our age-stratified analyses showed that urinary NO₃⁻ was positively associated with muscle strength in older adults, whereas no association was observed in younger and middle-aged adults, suggesting an age-dependent relationship. The mechanisms underlying this age-specific association are not fully understood, but several hypotheses can be proposed. It has been suggested that NO₃⁻ may influence muscle Ca²⁺ metabolism by enhancing calcium uptake and facilitating muscle contraction [36, 37]. Given that older adults exhibit reduced Ca²⁺ sensitivity compared to younger adults, and that the rate of muscle fiber force redevelopment depends on Ca²⁺ concentration [8], it is plausible that higher NO₃⁻ intake could enhance muscle contraction, particularly in populations with impaired contractile function, such as older adults. In contrast, since healthy younger and middle-aged adults generally maintain adequate muscle contraction capacity and Ca²⁺ sensitivity [8], it is unlikely that a significant association would be observed in this population. Additionally, another hypothesis can be considered. Foods rich in NO₃⁻ are predominantly vegetables [18], which also contain substantial amounts of antioxidant compounds [44, 45]. Antioxidants have been associated with better muscle strength, particularly in older adults [14, 15, 17, 46, 47], as this population experiences increased oxidative stress, which contributes to muscle strength decline [10]. To explore whether urinary nitrate may reflect intake of these foods, we conducted an additional analysis correlating urinary nitrate and the urinary nitrate-to-osmolality ratio with several nutrients. We found significant but modest positive correlations (r < 0.3) with nutrients typically derived from vegetables, such as fiber, vitamin C, and vitamin K (Supplementary Table 2). These findings suggest that urinary nitrate is at least partially determined by the intake of foods that are sources of these nutrients. Although the correlation coefficients were low, this outcome is not unexpected given the inherent limitations of 24-hour dietary recalls [35] and the fact that urinary nitrate reflects only about 65% of dietary nitrate intake [25, 26]. Therefore, we interpret these low correlations as consistent with the expected biological variability and methodological limitations, supporting the idea that urinary nitrate may serve as an indicator of vegetable- and nitrate-rich food intake. Nevertheless, caution is warranted, as direct measures of dietary nitrate intake were not available in the NHANES dataset.
To ensure that our findings were not confounded by urine concentration, given that dehydration may artificially elevate urinary nitrate levels by increasing solute concentration rather than reflecting true nitrate excretion, we conducted an additional analysis using the urinary nitrate-to-osmolality ratio. This approach accounts for variability in urine dilution and minimizes potential bias related to hydration status. Although the sample size was reduced due to missing osmolality data, the results were consistent with those obtained when urinary nitrate was analyzed alone, reinforcing that our findings are not driven by urine concentration. Moreover, this subanalysis yielded larger β coefficients, suggesting a stronger biological relevance of the observed association.
The present study has several limitations. Due to its cross-sectional design, causal inferences cannot be made and reverse causation cannot be excluded. In addition, the observed associations may still be influenced by residual confounding from unmeasured or imperfectly measured factors. However, we attempted to mitigate these concerns by adjusting the regression models for a comprehensive set of potential confounders. We observed a significant association between urinary nitrate and muscle strength only in older adults, but not in younger and middle-aged adults, suggesting that the association may be more evident in older age groups. However, this interpretation should be made with caution. In an additional analysis (data not shown) evaluating an interaction term (NO₃⁻ × age group), the interaction test yielded p for interaction = 0.089, indicating a borderline trend toward effect modification by age. Therefore, the interaction analysis does not provide strong statistical evidence that the association differs by age group, and we cannot exclude the possibility that the apparent age-specific findings reflect chance or residual confounding within the older-adult subgroup. Additionally, since this study used a publicly available dataset representative of the U.S. population, the findings cannot be generalized to other populations or to clinical populations. The beta coefficients observed in the present study were small, which warrants caution when considering the clinical relevance of the findings. However, it is important to note that, in our primary analyses, urinary NO₃⁻ was expressed in ng/mL, a small unit that likely contributes to the low magnitude of the estimated coefficients. Therefore, to facilitate interpretation and enhance the clinical meaningfulness of the estimates, we additionally analyzed urinary nitrate in µg/mL. Moreover, because urinary NO₃⁻ is not normally distributed, interpretation should focus mainly on the direction of the association rather than the absolute magnitude of the beta estimates. Another limitation is the lack of information regarding specific food sources contributing to urinary NO₃⁻ levels, which prevents extrapolation of the findings to particular foods. Moreover, establishing precise intake recommendations for NO₃⁻ sources based on these results is not feasible. While the findings suggest a potential benefit of NO₃⁻ intake for older adults, further research is needed to determine appropriate intake levels and to develop clear dietary guidelines. It is also important to note that our results are more reflective of acute NO₃⁻ consumption, as plasma NO₃⁻ levels typically peak within 1–2 h after ingestion and return to baseline within approximately 24 h [24], with about 65% excreted in the urine within 24–48 h [25, 26]; thus, the effects of chronic NO₃⁻ intake should be investigated in future studies. Despite these limitations, this study has notable strengths. We assessed NO₃⁻ intake using urinary levels, which serve as an objective biomarker and reflect approximately 65% of consumed NO₃⁻ [25, 26]. Additionally, our data are derived from a nationally representative U.S.A sample, enhancing the external validity of the findings within this population.
Conclusion
In conclusion, our study demonstrated a positive association between urinary NO₃⁻ levels and muscle strength in older adults, but not in younger and middle-aged adults, indicating an age-specific relationship. Longitudinal studies are needed to investigate the association between habitual chronic NO₃⁻ consumption and muscle strength in older adults and younger and middle-aged adults to clarify the clinical relevance of this relationship.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
No sources of funding were used to assist in the article preparation. EPO acknowledges the Brazilian National Council for Scientific and Technological Development (CNPq, Brazil, 307786/2022-6). RN acknowledges the Fundação de. Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). The authors also acknowledge CAPES for financial support.
Author contributions
DMM contributed to data interpretation, conducted statistical analysis, and wrote the manuscript; RN, MEFM and FMSB conducted statistical analysis, participated in data interpretation, and contributed to manuscript revisions; EPO wrote and reviewed the manuscript, and contributed to data interpretation. All authors reviewed and approved the final version of the manuscript.
Data availability
Data from the National Health and Nutrition Examination Survey (NHANES) are publicly available and can be accessed directly at (https://wwwn.cdc.gov/Nchs/Nhanes).
Declarations
Ethics approval and consent to participate
NHANES data collection protocols are approved by the ethics review board of the National Center for Health Statistics. Informed consent was obtained from all participants.
Informed consent
Informed consent was obtained from all individual participants included in the study.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Data from the National Health and Nutrition Examination Survey (NHANES) are publicly available and can be accessed directly at (https://wwwn.cdc.gov/Nchs/Nhanes).