Introduction

Progressive incremental testing (PIT) is an essential tool for evaluating and controlling training performance across three intensity domains in physical exercise: moderate, intense, and severe. Researchers use this framework to pinpoint two crucial metabolic transition points (MTPs) for both assessing and prescribing training regimens (Hogan, 2021; Vasconcelos et al., 2019). The aerobic threshold (AT), or threshold 1, indicates a shift toward greater aerobic energy contribution. Conversely, the respiratory compensation point, or threshold two, marks an increased lactate production and accumulation, demanding more from the anaerobic system to aid the aerobic system in energy production and maintenance (Jamnick et al., 2018). This synergy allows for sustained physical effort. Traditional methods to detect these points, however, rely on costly and invasive techniques such as gas analysis and blood lactate measurements, restricting personalized assessments (Machado et al., 2013; Quittmann et al., 2022).

Researchers have developed non-invasive techniques that simplify and reduce the costs of such evaluations. These include heart rate (HR) analysis in PIT, a method Conconi and colleagues have utilized to detect MTPs through HR fluctuations (Bodner & Rhodes, 2000; Pereira et al., 2017). Identifying the exercise intensity at these transition points is critical for trainers and exercise physiologists. Empirical evidence confirms that on a treadmill, PIT can accurately identify these points, with the first coinciding with the maximum rate of HR variation (Peserico et al., 2016), known as HRIP, which closely correlates with the aerobic threshold. The second point, termed HRDP, indicates the second lactate threshold and approximates the intensity of the maximum lactate balance (Cambri et al., 2008; De Assis Pereira et al., 2016)

Recently, nitrate (NO3-) has garnered attention for its vasodilatory properties, which improve athletic performance and enhance the bioavailability of nitric oxide (NO) (Garnacho-Castaño et al., 2018). Research has highlighted NO3-’s impact on the velocity, VO2, and HR of runners (Tan et al., 2018), but studies focusing on its influence on HR-defined MTPs are limited. Also, the reproducibility and correlation of peak velocity (Vpeak) with PIT performance in individuals taking NO3- supplements have not been adequately explored. Given its ease of use and low cost, Vpeak represents a viable tool for coaches to prescribe and monitor athlete training. We propose a novel approach: employing HR and its derivatives, HRIP and HRDP, along with Vpeak, to detect MTPs in lower-level amateur runners using acute NO3- supplementation through beetroot juice.

Methodology

Participants

This prospective, double-blind, placebo-controlled, crossover study received approval from the Ethics Committee in Research with Human Beings under Resolution CNE n. 466/12 and opinion approval number 3,663,376. All participants signed the Informed Consent Term (ICT). We selected fifteen amateur male runners from Varginha, Minas Gerais, based on specific inclusion and exclusion criteria. Thirteen completed the study. The sample power, calculated using Gpower 3.1, achieved a β value of 0.87. Inclusion criteria included being male, aged 20-35, engaging in more than two hours of training per week, training more than twice a week, having over two years of endurance running experience, familiarity with treadmill training, and participation in at least one 5 km running competition in the past five years. Exclusion criteria excluded runners with a 5 km test time over 25 minutes, chronic cardiovascular diseases, osteoarticular injuries in the past six months, use of any ergogenic aids or dietary supplements, inability to attend scheduled running tests, or injuries during the testing periods.

Study Design and Intervention

Participants were randomly assigned to either the placebo (PLA) group or the beetroot juice (BJ) group enriched with nitrate, in a double-blind arrangement by non-participating individuals. We administered the supplementation two hours before the tests, following International Olympic Committee (IOC) guidelines (Maughan et al., 2018). Each participant received either 70 ml of concentrated beet juice with ~6.4 mmol of NO3- (Beet IT; James White Drinks Ltd, Ipswich, UK) or a nitrate-depleted placebo containing 0.04 mmol NO3- (Beet IT; James White Drinks Ltd, Ipswich, UK). The design included a seven-day washout period between treatments. Figure 1 outlines the intervention protocol.

Participants were instructed to avoid consuming NO3- rich foods (green leafy vegetables, beets, and ultra-processed foods) and engaging in strenuous exercise 48 hours before testing. They were also advised not to use analgesics and muscle relaxants one hour before testing or use mouthwash.

Physical Assessment

On the first assessment day, we collected height and body mass data using a Welmy® scale with a stadiometer (model 110 FF, Santa Bárbara d’Oeste, Brazil). Adipose tissue (AT) was measured using a Bodymetrix® ultrasound device (model BX 2000), employing the Durnin and Womersley 4-skin-fold protocol (Durnin & Womersley, 1974).

Figure 1
Experimental design.

Progressive Incremental Testing (PIT)

Subjects performed an incremental test on an Embreex® treadmill (model 820 EXI), with speeds ranging from 1.2 to 20 km/h, after taking NO3- or a placebo. The protocol, proposed by Heck (Heck et al., 1985), began with a five-minute warm-up at four km/h without incline. The test, starting at eight km/h with 1.0 km/h increases every two minutes and a constant 1% incline, ended when subjects could no longer maintain pace despite encouragement or verbally indicated inability to continue. A maximum effort was defined as reaching at least 90% of HRMAX predicted by Tanaka’s formula (Tanaka et al., 2001):

We recorded HR at the end of each stage using a Garmin® HR monitor (model Fênix 3). HRMAX was predicted using Tanaka’s formula (Tanaka et al., 2001):

Identification of the Heart Rate Transitions Points

We plotted HR and velocity data every two minutes to identify HRIP and HRDP using Cambri’s mathematical model (Cambri et al., 2008), which involves adjusting HR-velocity values with a third-degree polynomial function and a first-degree linear equation.

Statistical Analysis

Statistical analyses were conducted using SPSS® version 25.0 and graphs were plotted using Prism® version 8.0. We employed the Shapiro-Wilk test to assess data normality. T tests for two non-independent samples assessed means equality and Levene’s test assessed variance equality, with significance set at p<0.05. We calculated the variation delta (Δ) and used the Cohen test (d) to measure effect size, classifying it as trivial (<0.35), small (0.35-0.80), moderate (0.80-1.50), and large (>1.5) for recreationally trained individuals (Rhea, 2004).

Results

Our study involved 13 participants over a period of four consecutive weeks, with details of the sample presented in Table 1.

Supplementation with either placebo or nitrate showed no significant difference in HRMAX, with PLA recording 182.77±4.83 bpm and BJ 181.54±6.07 bpm (d=0.013; p=0.573) (Fig. 2A). Similarly, MTP data revealed no significant changes in the HRIP or the HRDP after supplementation. The HRIP measurements were PLA: 143.23±6.75 bpm versus BJ: 139.53±7.99 bpm (d=0.013; p=0.252) (Fig. 2B), and HRDP measurements were PLA: 151.46±6.09 bpm versus BJ: 150.23±6.23 bpm (d=0.038; p=0.508) (Fig. 2C). Furthermore, the %HRMAX at the HRIP and HRDP post-supplementation also indicated no significant differences (PLA: 78.42 ± 4.25% vs. BJ: 76.92 ± 4.80%, d= 0.029; p=0.839 and PLA: 82.91 ± 3.75% vs. BJ: 82.81 ± 3.80%, d= 0.01; p=0.067, respectively) (Figs. 2D and 2E).

Figure 2
Variable’s heart rate in the progressive test.

Supplementation with placebo or nitrate did not significantly affect Vpeak (PLA=17.83 ± 1.98 km/h-1 vs. BJ=18.53 ± 2.09 km/h-1, d=0.031; p=0.301) (Fig. 3A). Likewise, the velocities at the HRIP and HRDP post-supplementation showed no significant differ-ences (PLA=10 ± 0.40 km/h-1 vs. BJ=10±0.89 km/h-1, d=0.013; p=0.562 and PLA=12.23 ± 1.69 km/h-1 vs. BJ=12.92 ± 1.93 km/h-1, d=0.038; p=0.340, respectively) (Figs. 3B and 3C). Additionally, the % Vpeak at the HRIP and HRDP after supplementation also demon-strated no significant changes (PLA=56.63 ± 5.89 % vs. BJ=55.23 ± 6.04%, d= 0.015; p= 0.598 and PLA=68.91 ± 8.91% vs. BJ=69.89 ± 8.05%, d=0.04 p= 0.295, respectively) (Figs. 3D and 3E).

Figure 3
Variable’s Vpeak in the progressive test.

Discussión

To predict functional capacity for exercise, we identified MTPs that increase anaerobic and/or aerobic energy production involvement. Our study uniquely analyzed the effects of NO3- supplementation on HRMAX, HRIP, HRDP, and Vpeak in the PIT of Comparative analysis proves challenging as current literature scarcely addresses these variables at maximum effort and their thresholds following acute NO3- supplementation, leading to ongoing debates and controversies about the occurrence of these variables and the optimal protocol for their identification (Jones & Jones, 2022; Poole et al., 2021). Although no significant differences emerged between groups, we noted a trend towards performance enhancements in the nitrate group, reflected in notable individual performance gains.

Our findings for the first threshold from the HRIP, despite lacking significant differences, indicate that the supplemented group reached the aerobic threshold at a lower HR than the placebo group, suggesting less cardiac effort, improved tolerance in the heavy domain, and enhanced buffering. Ghiarone and colleagues also reported similar HR behaviors in identifying transition points in 13 young athletes during progressive athletic gait testing before and after training sessions (Ghiarone et al., n.d.; Nummela et al., 2007). The pre-training HRIP values, particularly at kilometre 7.00 (n=8-5.31 km·h-1; 125 bpm), and the post-training HRIP values (n=12-5.46 km·h-1; 125 bpm) mirrored ours (Camargo et al., 2017). Couto et al. (2013) found significantly higher HRIP values in 20 university students during cycle ergometer testing compared to our 13 participants (126 bpm±12). While we observed no velocity differences between groups, the BJ Group reached the aerobic threshold at higher velocities, indicating effective adaptive training, increased lactate tolerance and removal, and reduced bodily stress (Jones & Jones, 2022). Percentage-wise, the supplemented group demonstrated lower maximum speed percentages, suggesting reduced energy consumption and O2 use. Cambri et al., (2008) observed similar speeds at the final HRIP among 20 university students undergoing maximum progressive treadmill testing (10.9±1.8).

For the second threshold from the HRDP, our study also recorded lower HR values in the supplemented group compared to the placebo group, though these differences were not statistically significant. Moreover, the velocity sustained by the supplemented group surpassed that of the placebo group. We attribute the enhanced tolerance to the severe domain, observable through lower HR and higher speeds, to NO3- induced vasodilation, improved buffering, and H+ ion absorption, alongside decreased oxygen consumption (McQuillan et al., 2017; Tan et al., 2018). This improved tolerance ultimately brings athletes’ HRDP closer to their VO2MAX, offering practical benefits.

HRMAX frequently serves as a physiological marker to gauge effort intensity in sports, including during progressive tests and assessments of HR behavior across various exercise intensities (Vobejda et al., 2006). In this study, the HRMAX and its percentage values at transition thresholds did not show significant differences. However, we noted a trend towards positive behavior in the BJ group and detrimental behavior in the PLA group. The lower HRMAX values and those at transition points support our theory that NO3- enhances threshold tolerance and, thereby, performance (Ghiarone et al., 2017).

Researchers observed HR behavior among twenty university students during maximal progressive treadmill testing. They identified the HR transition points; the initial phase’s HR inflection point was 130±8 (64.4±5.3%), and the final HR reached 195±10 bpm (97.4±3.4%). These results align with the established literature on the curvilinear relationship between HR and work intensity, and the presence of frequency transition points at both the beginning and end of the curve (Cambri et al., 2008). A separate study investigated heart rate deflection in ten runners during a maximal incremental load running test without supplementation. This study recorded higher HR values at the threshold (174.4 ±6.1 bpm) and slightly lower velocities at the HR threshold (11.4 ±0.8 km/h-1) compared to our findings (Denadai et al., 2004).

Vpeak, the maximum speed achieved in an incremental test, effectively predicts endurance running performance and indicates training prescription efficacy (De A. Manoel et al., 2022; Machado et al., 2013). However, our study did not compare Vpeak with and without acute NO3- supplementation, a notable limitation. Despite substantial evidence supporting Vpeak as a better performance predictor than lactate threshold- demonstrated in a study of 43 marathon and ultramarathon runners over distances ranging from 10 to 90 km (Alvero-Cruz et al., 2020) -our data did not confirm NO3- interference in Vpeak. Nevertheless, other studies have established Vpeak as an effective performance evaluator in short 3- and 5-km tests (Borszcz et al., 2018; De A. Manoel et al., 2022; Denadai et al., 2004).

In this study, Vpeak did not significantly differ between the supplemented and placebo groups. However, we observed a trend where participants in the BJ group maintained higher speeds (+0.7 km/h-1) and showed a lower percentage at the HRIP, suggesting less fatigue at transition points, thereby enhancing performance. These observations, while not statistically significant, support the hypothesis that supplemented individuals can capture and metabolize more oxygen during the aerobic to anaerobic transition, resulting in greater endurance, reduced acidosis, and decreased cardiac effort at higher intensities (Hogan, 2021; Jones et al., 2019; Vasconcelos et al., 2019). Additionally, NO might enhance oxygen transport to muscle fibers (Rokkedal-Lausch et al., 2019).

A study involving swimmers showed that nitrate-rich beet juice supplementation reduced energy costs and increased workload at the anaerobic threshold (Pinna et al., 2014). The results indicated a significant increase in workload at the anaerobic threshold compared to baseline values (6.3 ± 1 to 6.7 ± 1.1 kg), and a reduction in oxygen costs during tests post-supplementation (1.7 ± 0.3 kcal·kg−1·h−1 to 1.9 ± 0.5). These findings suggest that the nitrate group achieved a prolonged duration to reach the second threshold, highlighting the potential benefits of supplementation (Pinna et al., 2014)

Machado et al. (2013) investigated the relationship between 5 km running performance and Vpeak in untrained men and women using an incremental treadmill test. They found lower Vpeak values (12.2 ± 1.8 km·h-1) compared to ours, yet confirmed Vpeak as a strong indicator of training prescription efficacy, with significant performance differences between male and female participants (P < 0.001) (Machado et al., 2013) De A. Manoel et al., (2022) also identified Vpeak and velocity as markers of VO2MAX and effective training prescription indicators. They studied the impact of 4 weeks of Vpeakbased or vVO2MAX-based training on 14 moderately trained runners, showing that both metrics significantly correlated with 10 km performance pre- and post-training (Vpeak -0.97 vs. -0.87 and vVO2max -0.95 vs. -0.94) (De A. Manoel et al., 2022).

Garrett et al. (2023) demonstrated that Vpeak could serve as a cost-effective method to measure neuromuscular fatigue by comparing its sensitivity during a submaximal running test and a countermovement jump test among 20 semi-professional soccer players (Garrett et al., 2023). In contrast, Figueiredo et al., (2021) assessed Vpeak and Vcrit as predictors of 5 km performance in twenty recreational runners, finding a higher correlation and predictive power for Vcrit (13.7 ± 1.1 vs. 12.1* ± 1.4 km·h-1, P < 0.001) (Figueiredo et al., 2021).

We acknowledge limitations in our study, such as using HR as an indirect measurement method and not measuring serum nitrate and nitrite levels. However, our findings highlight the utility of Vpeak as a practical, low-cost variable for evaluating runners. This study emphasized the importance of small adjustments in effort and velocity for performance enhancement through training adaptations in athletes.

Conclusions

Nitrate supplementation does not significantly affect the performance of amateur long-distance runners during PIT. Despite the lack of significant differences in parameters, the observed improvements with nitrate supplementation could be relevant in practical settings. The findings associated with HRIP and HRDP indicate enhancements, such as reduced cardiac effort at MTPs and increased Vpeak following nitrate supplementation. These results imply that NO-3 supplementation potentially enhances competitive performance by improving tolerance to heavy and severe exercise domains and increasing speed. Therefore, incorporating nitrate supplementation into the training programs and diets of these athletes may prove beneficial

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