The Effect of One-Week Spinach and NBS Superfood Supplementation on Interleukin-6, Superoxide Dismutase, and Malondialdehyde Levels after Repeated Bouts of Wingate Test in Trained Men

doi:10.21203/rs.3.rs-4915469/v1

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The Effect of One-Week Spinach and NBS Superfood Supplementation on Interleukin-6, Superoxide Dismutase, and Malondialdehyde Levels after Repeated Bouts of Wingate Test in Trained Men

https://doi.org/10.21203/rs.3.rs-4915469/v1

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Background: Consuming nutritional supplements for improving athletic performance has always been considered an ergogenic aid. However, there is limited information on the efficacy of this strategy for enhancing antioxidant capacity in response to strenuous exercise with repeated nature. Therefore, this study investigated the effect of one week of spinach and NBS superfood supplementation on superoxide dismutase, malondialdehyde, and interleukin-6 in response to repeated bouts of Wingate test.

Methods: Fifteen trained men voluntarily participated in this within-subject and counter-balanced study. Participants were exposed to three different conditions including 1) seven days of raw spinach supplementation (daily dose of 70 g); 2) seven days of NBS superfood supplementation (daily dose of 10 g); and 3) the control condition. One day after the supplementation period, participants came to the lab and performed 3 bouts of 30-second Wingate test with 4-minute rest intervals. Before the supplementation and 24 hours after performing the Wingate, test blood samples were taken in each condition to measure serum levels of superoxide dismutase, malondialdehyde, and interleukin-6. One week of wash-out was applied between the conditions. Two-way repeated measures ANOVA were used for statistical analysis.

Results: The results showed that at post-test measurement the serum level of interleukin-6 was significantly lower under the spinach and superfood conditions (p=0.001, p=0.003, respectively) compared to the control. The serum level of superoxide dismutase under the spinach (p=0.035)and superfood (p=0.01) conditions was significantly higher compared to the control at post-test measurement. Also, at post-test measurement, the serum level of malondialdehyde was significantly lower under the spinach (p=0.001)and superfood (p=0.017) conditions compared to the control.

Conclusion:According to the results of the present study, it seems that seven days’ supplementation of either raw spinach or NBS superfood could positively affect the inflammatory responses to repeated anaerobic all-out activities. Therefore, these supplementation strategies might be considered ergogenic aids and used by athletes before participating in repeated strenuous activities.

Nutritional Supplementation

Anaerobic Activity

Inflammatory Response

Ergogenic Aid

High-intensity exercise increases reactive oxidative species (ROS) levels in skeletal muscles, resulting in inflammation and force loss[1]. Such activities disrupt the balance between oxidative stress and anti-oxidative defense and allow for the production and accumulation of ROS in the mitochondrial respiratory chain and other metabolic pathways [2, 3]. ROSs are highly reactive molecules that are produced during cellular metabolic activities and can react with biological macromolecules in cells such as carbohydrates, nucleic acids, lipids, and proteins due to the presence of unpaired electrons in their structure and disrupt their normal function [4, 5]. In fact, oxidative stress indicates an imbalance between oxidants and the body's antioxidant capacity. This imbalance and the production and accumulation of ROS leads to disruption of signaling and oxidation of essential biomolecules such as lipids and proteins in cells [6]. Intense exercise also causes oxidative stress in human blood cells and leads to lipid peroxidation, DNA damage, mitochondrial abnormalities, and protein oxidation [7, 8]. Subsequently, the activity of antioxidant enzymes including superoxide dismutase (SOD) increases in order to neutralize free radicals and prevent harmful effects of ROS [9]. Following this process, the concentration of antioxidant enzymes such as superoxide dismutase in blood decreases [10]. It has been shown that one session of high-intensity resistance training increases plasma malondialdehyde (MDA) concentration [11]. It has also been demonstrated that intensive physical exercise programs increase ROS and MDA levels, leading to inadequate recovery, muscle damage, and decreased physical performance [12].

Since oxidative stress and inflammation are associated with fatigue and disturbance recovery after intense exercise, research has focused on nutritional strategies aimed at reducing these effects [13]. A number of studies have evaluated the effects of antioxidant supplements before ultramarathon or triathlons of iron men [14]. For example, it has been reported that supplementation with coenzyme Q, melatonin, or other antioxidants before intense exercise reduced oxidative stress, modulated inflammation, and reduced subsequent muscle damage [15]. For this reason, consuming foods with antioxidant properties has been deemed an effective strategy to neutralize the risk of harmful oxidants and maintain this balance [16, 17]. Spinach is one of the most important antioxidant-containing food plants, which has a considerable amounts of nitrate, carotenoids, and flavonoids and is a good source of vitamins A, B3, and C and minerals such as potassium, calcium, magnesium, and iron [18].

Spinach has the highest amounts of alpha-lipoic acid among plant sources [19]. Alpha-lipoic acid is considered as a micronutrient and a powerful antioxidant which has high antioxidant capacity and contributes to mitochondrial activity and has a positive effect on exercise performance [20]. Alpha-lipoic acid is an essential coenzyme that is important for maintaining energy homeostasis in mitochondria [21]. Consumption of vegetables such as spinach containing nitrate can improve athletic performance by improving cardiorespiratory endurance followed by postponing fatigue [22, 23] and improving aerobic performance in less-than-moderate exercise intensities[24]. In this regard, Rouhi et al., showed that taking spinach supplements in healthy trained men after two half marathons increased total antioxidant capacity (TAC) and decreases MDA as an indicator of oxidative stress [25]. In another study, the effect of red spinach supplementation on athletic performance and cycling time in recreational active men and women was assessed. The results revealed that a 7-day red spinach supplementation significantly reduced the time of performing a cycling time-trial task [26].

Another natural nutrient that can be used as a supplement is the NBS superfood supplement. The recently produced Bio-shield Superfood NBS Nutrition is a new, organic, and healthy herbal supplement. This herbal supplement contains macro and micro molecules, various vitamins including B1, B2, B3, B5, B6, B9, C, D, K, E, and the minerals such as magnesium, potassium, phosphorus, manganese, calcium, iron, copper, zinc, omega-3, omega-6, omega-9, and other substances [27]. Although NBS has been shown to have many potential health benefits, there are currently limited human studies on the effectiveness of NBS superfood supplements. In one study, NBS superfood supplementation in mice significantly increased the number of white blood cells and neutrophils and helped the immune system respond to pathogens [28]. In another study on the liver of mice, the results of biochemical blood tests in mice showed that rats supplemented with superfood returned to normal liver parameters [27]In a human study conducted on COVID-19 patients, the findings showed that the use of NBS superfood had various beneficial effects on the severity of COVID-19. Reduction of CRP, ESR, D-Dimer, LDH, SGOT, SGPT, body temperature, IL6, and TNF-a, as well as increased blood oxygen levels were notable effects of NBS. In addition, the results showed that the use of NBS superfood supplement reduced mortality rates in COVID-19 patients and significantly reduced the length of hospitalization in COVID-19 patients [29].

In most of the previous studies investigating the effects of nutritional supplements on oxidative stress, aerobic exercise or resistance exercise has been used as the training protocol, nevertheless, many sporting activities involve short bouts of maximal or all-out effort ranging from a few seconds to less than one minute interspersed with periods of sub-maximal activities in between, also known as anaerobic activity [30]. Anaerobic activities in which repeated sprints with longer duration (15 to 90 s) must be performed, known as speed-endurance or speed-strength activities, are mostly rely on glycolytic system as the source of energy [31]. In such activities, metabolic perturbations lead to a high accumulation of metabolic by-products and an increase in oxidative stress [32]. Unfortunately, despite the relevance of using nutritional supplements to cope with the anaerobic-induced oxidative stress, this area of study has almost been overlooked and there is a dearth of information in this regard.

Therefore, considering the antioxidant and anti-inflammatory properties of spinach and NBS superfood, and limited research on the efficacy of such nutritional supplements on the oxidative stress resulted from anaerobic performance with repeated nature, we investigated the effect of a 7-day spinach or NBS superfood supplementation on IL-6, SOD, and MDA after performing repeated bouts of the Wingate test. We hypothesized that 7-day spinach or NBS superfood supplementation will decrease interleukin 6 and malondialdehyde; and increase superoxide dismutase after performing repeated all-out (30-s Wingate test) activities.

Subjects

Fifteen healthy and active men voluntarily participated in the study. The subjects characteristics are presented in Table 1. The sample size was calculated using G*Power software (Version 3.1.9.2, Kiel, Germany) software as follows: test family = F tests; Statistical test = ANOVA: Repeated measures, within factors; α error probability = 0.05; power (1-β err prob) = 0.80; Effect size f = 0.35, number of groups = 1, number of measurements = 3, Correlation among repeated measures = 0.5, and Non-sphericity ɛ correction = 1. Accordingly, 15 subjects were appropriate as the sample size for the present study. The inclusion criteria were (a) age range of 18 to 30 years, (b) Body Mass Index (BMI) 18.5 to 24.9 kg/m2, and (c) history of familiarity and performing anaerobic exercises. The exclusion criteria were (a) developing any type of cardiovascular, pulmonary, and metabolic disease, (b) history of seizures, epilepsy, or other types of neurological diseases, (c) smoking and alcohol use, (d) voluntary withdrawal of subjects from the study, (e) suffering from a variety of acute diseases in the executive phase of the research, and (f) not participating in any of the sessions of the executive phase of the research. This study was approved by the Ethics Committee of Razi University (IR.RAZI.REC.1403.013) and registered in the Iranian Registry of Clinical Trials (IRCT id IRCT20210617051606N5; Registration Date: 00/00/2022). All participants gave their written informed consent to the experimental design of the study.

Table 1

**General characteristics of participants**
Variables	Mean ± SD (n = 15)
Age (years)	22.1 ± 3
Weight (kg)	60.71 ± 2.7
Height (cm)	176.89 ± 1.4
BMI (kg/m2)	21.14 ± 1
Body Fat (%)	17.6 ± 3.1
Fat Mass (kg)	12.55 ± 4.9
RT experience (years)	4.2 ± 1.8

Experimental Procedure

First, the subjects participated in a familiarization session to be acquainted with whole stages of the research, performing the tests, and measuring the study variables. The anthropometric characteristics of the subjects were also measured in the same session. To avoid any abnormal reaction to spinach and NBS superfood consumption, subjects were given small doses of spinach and superfood. None of the participants reported abnormal reaction within 48 h after consuming spinach and NBS superfood. Then, the subjects were randomly assigned to three different conditions including: 1) seven days of spinach supplementation; 2) seven days of NBS superfood supplementation; and 3) control condition. Seven days of wash-out were applied between the conditions. The experimental procedure for the subjects was as follow, the first blood sample was taken before being exposed to the experimental condition as the pre-test for the respective condition (spinach, NBS superfood, or control). Then, the subjects were exposed to 7 days of either spinach or NBS superfood supplementation, or being under control condition. 24 hours after the end of each condition, the subjects came to the lab an performed 3 bouts of 30-s Wingate test with 4 minutes of recovery between each bout. 24 hours after performing the Wingate test, the second blood sample were taken as the post-test for the respective condition. Then, the subject had 7 days of wash-out period and then, the same procedure was applied for the second and third condition. The amount of use of spinach or the dose of spinach for each subject was 70 grams of fresh raw spinach packed per day before bedtime. The serving size was calculated using the USDA National Nutrient Database was standard reference [33]. The NBS superfood supplement dose was 10 grams per day before bedtime [34]. A 24-hour paper-based dietary recall was applied by a nutrition expert (through an interview with each participant) before the first experimental session and participants were instructed to follow the same diet 24 hours before the next two experimental sessions (the sessions they performed the Wingate test). Moreover, to avoid any effects of circadian rhythm on the study variables, each subject came to the laboratory at the identical time of the day in a laboratory-controlled ambient condition (19–22°C; 50–60% relative humidity) in all experimental sessions. The whole experimental procedure has been depicted in Fig. 1.

Session 1

Familiarization with the whole Experimental Procedure

Session2,3 and 4 3 bouts of 30-s Wingate test under 3 different conditions
the first blood sample was taken before being exposed to the experimental condition
the subjects were exposed to 7 days of either spinach or NBS superfood supplementation, or being under control condition
Baseline of blood sample (pre -Test)
3 bouts of 30-second Wingate test with 4-minute active recovery
Supplementation Randomly defined
Repetitive Wingate Test
the second blood sample (post -Test)
24 hours after performing the Wingate test

Blood Sampling and Sample Measurement

Before receiving the supplement and starting each condition, the subjects attended the laboratory and a blood sample of 6 ml of Cubital fossa was taken. It should be noted that all blood samples were taken in fasting mode by laboratory experts of the hospital. Another sample was taken 24 hours after the training session (three bouts of Wingate test). Blood samples were collected in tubes containing anticoagulant EDTA (ethylene diamine tetra acetic acid). Blood plasmas were stored at -80°C until further tests were performed. To separate the serum, the serum was placed at room temperature for 20 minutes to clot, then blood samples were centrifuged at 3000 rpm for 15 minutes and were used to measure blood factors using standard kit. The levels of IL6, MDA, SOD was measured by kits (96/48 Tests) ZellBio GmbH (Germany) CAT No، photometry with a spectrophotometer with a wavelength of 546 n.

Spinach and NBS Superfood Supplementation

The amount of use of spinach or the dose of spinach for each subject was 70 grams of fresh raw spinach packed per day before bedtime. The serving size was calculated using the USDA National Nutrient Database was standard reference [33]. The NBS superfood supplement dose was 10 grams per day before bedtime. NBS superfood supplement is known as a herbal supplement that has therapeutic and sports properties. This herbal supplement is basically prepared from grains (mostly wheat), which have been enriched and improved the properties of this supplement. In terms of nutritional value, this organic supplement contains 25% protein, 50% carbohydrates, 15% fiber, 10% omega-3 and omega-6 and other vitamins. The daily dose of superfood for each subject was considered to be 10 grams; According to reliable scientific sources, it is suggested that 4.5 grams [34]. should be consumed daily for each non-athlete and healthy person, and this dose can be changed in different people with different physical characteristics and duration of consumption up to 20 grams per day.

Repeated anaerobic performance (Wingate Test)

The subjects performed three bouts of the 30-s cycling all-out Wingate test with 4 min of active recovery on a cycle ergometer (Ergomedic 894E, Monark Sports and Medical, Stockholm, Sweden). The bike settings (saddle height) were kept the same in all experimental sessions, according to each preference set in the familiarization session. A standard 5-minute warm-up was performed, consisting of pedaling with a 2% resistance of total body weight and performing 3 sprints of 5s at minutes 2, 3, and 4 with a 6.6% resistance of total body weight. After the warmup and before starting the test, a 3-min passive recovery was applied, and then, with the command “Go”, the participants started pedaling as fast as possible for 30s against a constant load equal to 7.5% of each participant’s body mass. After each bout of the Wingate test, the participants performed 4 min of active recovery on the bike with 30–40 W at 50 rpm. During the last 10 s of the recovery, the participants were informed to be ready for the next bout and the last 3 s of the recovery phase were counted down by the experimenter to inform the participants of the start point of the next bout of the Wingate test. Strong verbal encouragement was provided during each bout of the Wingate test. The Peak power (PP: highest power output achieved during the 30 s test) and mean power (MP: average power calculated for the complete test duration) were obtained in each bout. The fatigue index was subsequently calculated [FI = (Peak Power - Minimum Power) / (Peak Power × 100)] [35, 36].

Statistical Methods

Data are presented as the mean ± standard deviation (M ± SD). The normal distribution of each data set was evaluated by the Shapiro-Wilk normality test. Two-way repeated measures ANOVA (3 × 2 factorial design; 3 stimulation conditions and 2-time points) was used to analyze IL-6, SOD, and MDA at pre- and post-test. Bonferroni post hoc test was used for the pairwise comparisons. In case of a violation in the assumption of sphericity, the Greenhouse-Geisser epsilon correction was applied. Partial eta squared (ɳ²_p) was used as a measure of the effect size for the ANOVAs and interpreted as small (0.01–0.059), medium (0.06 to 0.139), or large (≥ 0.14). The statistical analyses were performed using SPSS 27 (SPSS Inc., Chicago, IL, USA) and p˂0.05 was adopted.

The overall results of the study variables are presented in Table 2.

Table 2

Mean values of the study variables at specifed time points under 3 different conditions
Variables	Experimental Conditions Mean ± SD (n = 15)
	Super Food		Spinach		Control
	pre	post	pre	post	pre	post
IL-6	3.68 ± 1.16	11.22 ± 1.75	3.77 ± 1.28	5.86 ± 1.39	4.4 ± 1.41	18.03 ± 6.58
SOD	26.75 ± 14.4	36.16 ± 10.90	28.46 ± 9.51	37.53 ± 12.04	26.83 ± 7.81	27.25 ± 7.24
MDA	3.22 ± 1.6	2.3 ± 0.23	2.85 ± 0.6	2.24 ± 0.16	2.14 ± 0.5	2.68 ± 0.35

The results showed significant main effects of condition (F_{(1.1, 16.6)} = 43.43, p ˂ 0.001, ɳ²_p = 0.756), time (F_{(1.0, 14)} = 103.3, p ˂ 0.001, ɳ²_p = 0.881), and also “condition × time” interaction (F_{(1.1, 16.5)} = 35.43, p ˂ 0.001, ɳ²_p = 0.717) on the IL-6. Pairwise comparisons revealed that the IL-6 was significantly lower at post-test in spinach and NBS superfood conditions compared to the control (p ˂ 0.001; p = 0.003, respectively). The results also demonstrated that the IL-6 was significantly lower at post-test in the spinach condition than in the NBS superfood condition (p ˂ 0.001) (Fig. 2).

There were significant main effects of condition (F_{(2, 26)} = 19.5, p ˂ 0.001, ɳ²_p = 0.600), time (F_{(1, 13)} = 23.1, p ˂ 0.001, ɳ²_p = 0.640), and also “condition × time” interaction (F_{(1.2, 16.4)} = 18.38, p ˂ 0.001, ɳ²_p = 0.586) on the SOD. Pairwise comparisons indicated that at post-test, the SOD was significantly higher in the spinach (p ˂ 0.001) and NBS superfood (p ˂ 0.001) conditions compared to the control condition. No significant difference was observed between the spinach and NBS superfood conditions; Fig. 3.

MDA

Our results demonstrated a significant “condition × time” interaction effect (F_{(1.1, 14.9)} = 7.04, p = 0.015, ɳ²_p = 0.351) on the MDA, while no significant main effects of condition (F_{(1.3, 16.9)} = 0.96, p = 0.39, ɳ²_p = 0.069) and time (F_{(1, 13)} = 4.61, p = 0.051, ɳ²_p = 0.262) were seen on the MDA. Pairwise comparisons revealed that the MDA was significantly lower in the spinach (p ˂ 0.001) and NBS superfood (p = 0.017) conditions compared to the control condition in post-test; Fig. 4.

In this study, the results showed that one-week supplementation, 70 g daily of raw spinach and 10 grams daily of NBS superfood, controlled inflammation caused by three bouts of 30-second Wingate test. The findings of the present study showed that three sets of 30-second Wingate test are associated with an increase in inflammation and oxidative stress, which is also proven by previous studies. These studies have shown that following strenuous and frequent physical activity, ROS is produced and accumulated and contributes to inflammation in the body [36]. Continued strenuous and repeated physical activity over the long term can impair some immune cell functions due to free radicals [37]. It has been shown that increasing levels of IL-6 during exercise induces inflammatory response in the body, and inhibition of IL-6 signaling controls inflammatory responses [38].

As a result of intense exercise, interleukin-6, which is secreted from white blood cells, increases in the blood. In fact, an excessive increase in interleukin-6 means inflammation [38, 39]caused by disease or vigorous physical activity[40, 41]. In the present study, data obtained from interleukin-6 levels in the control condition after 24 hours of the three-stage Wingate test showed that three Wingate tests increased the level of interleukin-6 in the blood.

In our study, interleukin-6 levels decreased in the spinach and superfood supplementation conditions after 24 hours of the three-stage Wingate tests. In a study of corona patients, the results showed that taking 4.5 grams of NBS superfood supplements daily for 14 days reduced interleukin-6 levels.

No research has been done on spinach and its effects on interleukin-6, but in our study, the mechanism of spinach's possible effect on interleukin 6 seems to be related to its antioxidant and anti-inflammatory properties. The data from our study showed that the serum levels of superoxide dismutase increased in spinach and superfood supplementation conditions after 24 hours of the three bouts of Wingate test. Due to intense physical activity and subsequent ROS production, the activity of antioxidant enzymes such as superoxide dismutase (SOD) increases in order to neutralize the harmful effects of ROS [10] which leads to a decrease in the concentration of antioxidant enzymes such as superoxide dismutase in the blood [9]. Therefore, since superoxide dismutase prevents the accumulation of ROS in the body and on the other hand, the accumulation of ROS contributes to inflammation, it can be inferred that it is probably part of the mechanism of reduction effect of spinach on interleukin-6 through the antioxidant properties of spinach and increasing the superoxide dismutase enzyme in the body.

Previous studies on spinach have less evaluated the antioxidant properties of spinach. In a study on spinach, it was found that 14 days of spinach with a dose of 1 mg/1kg body weight increased antioxidant capacity [26]. In the present study, it seems that the increase in serum levels of superoxide dismutase in spinach condition is related to antioxidant properties of spinach; spinach has the highest amount of alpha-lipoic acid [20] as a potent antioxidant [19, 26]. On the other hand, studies on NBS superfood supplement have been more medical and therapeutic. There have not yet been complete human studies to evaluate the ergogenic and potential properties of this supplement. However, NBS superfood supplements have been proven to contain high levels of vitamin C and E [27]. It seems that in the present study, the increase in serum levels of superoxide dismutase in NBS superfood supplementation conditions is related to these antioxidant substances in NBS superfood.

The data of the present study showed that serum levels of malondialdehyde decreased in spinach and NBS superfood supplementation conditions after 24 hours of running three bouts of Wingate test. Past research has proven that strenuous physical activity is associated with increased plasma concentrations of malondialdehyde (MDA) (a marker of oxidative stress, or ROS) [11]. with increased levels of MDA leading to inadequate recovery, muscle damage, and decreased physical function [12]. Various studies have been done regarding malondialdehyde and exercise that have shown that increased levels of malondialdehyde after strenuous exercise indicate oxidative stress; in fact, due to intense activity, oxygen consumption by cells increases, which leads to accumulation of free radicals in the cell membrane of tissues, which if not controlled, leads to fatigue and damage to the mitochondrial membrane of cells, which disrupts the body's ATP production. Therefore, consumption of foods with antioxidant properties is of great importance in order to prevent free radical accumulation in mitochondrial membrane [42]. According to the data obtained in the present study, it seems that the mechanism of reducing effect of spinach and superfood NBS on malondialdehyde levels is related to their antioxidant properties. In fact, malondialdehyde is an indicator of oxidative stress which is considered as a harmful factor for health and activity and athletic performance; so, it seems that the cause of the decrease in malondialdehyde in the present study in the condition of spinach and NBS superfood supplementation is related to the beneficial antioxidant properties of these two plant foods.

In general, according to the results of this study, it can be concluded that consumption of spinach or superfood supplement can be effective in improving physiological indices after repeated activities of maximum anaerobic activities and can be used as a useful nutritional strategy for promoting athletes' performance and health in fields with repetitive anaerobic nature.

SOD Superoxide dismutase

MDA Malondialdehyde

IL6 Interleukin 6

NBS Nutrition Bio-shield Superfood

ROS Reactive oxygen species

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Razi University (IR.RAZI.REC.1403.013) and registered in the Iranian Registry of Clinical Trials (IRCT id IRCT20210617051606N5; Registration Date: 00/00/2022). Informed consent was obtained from all subjects before participation in the study.

Consent for publication

Not Applicable.

Competing interests

All authors declare that they have no competing interests.

Author details

Exercise Metabolism and Performance Lab (EMPL), Department of Exercise Physiology, Faculty of Sport Sciences, Razi University Kermanshah, Iran.

Funding

This study did not receive funding from any public or commercial sector.

Author Contribution

MMSH, MA, EA, conceptualized and designed the study. MMSH, MA, EA conducted the experiments. MMSH, MA participated in the formal analysis. MA wrote the original draft of the manuscript. MMSH, MA, EA, reviewed and edited the manuscript All authors approved the final version of the manuscript. MA supervised the project.

Acknowledgements

All authors express our sincere gratitude to the participants for their commitment during the study.

Data Availability

The data generated and/or analyzed during the current study are available from the corresponding author or reasonable request.

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The Effect of One-Week Spinach and NBS Superfood Supplementation on Interleukin-6, Superoxide Dismutase, and Malondialdehyde Levels after Repeated Bouts of Wingate Test in Trained Men

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Version 1

Abstract

Figures

Background

Methods

Subjects

Experimental Procedure

Blood Sampling and Sample Measurement

Spinach and NBS Superfood Supplementation

Repeated anaerobic performance (Wingate Test)

Statistical Methods

Results

MDA

Discussion

Conclusion

Abbreviations

Declarations

Author details

Funding

Author Contribution

Acknowledgements

Data Availability

References

Additional Declarations

Status:

Version 1