Sex-Based Differences at Ventilatory Thresholds in Trained Runners

Abstract

Objective: This study aimed to compare trained male and female athletes regarding physiological, perceptual, and performance variables at ventilatory thresholds (VT₁ and VT₂). Methods: Twenty-four male and nineteen female trained runners (age: 27.9 ± 6.4 vs. 24.4 ± 4.4 years; body mass: 61.8 ± 4.3 vs. 52.6 ± 4.1 kg; height: 174.6 ± 5.8 vs. 165.0 ± 5.0 cm for males and females, respectively) performed a graded exercise test to exhaustion on a treadmill. During the test, oxygen consumption, respiratory exchange ratio, running power output, heart rate, muscle oxygenation, and rate of perceived exertion were analyzed. Sex differences were evaluated with an unpaired-samples t-test. Results: Males exhibited significantly higher respiratory exchange ratios (0.87 ± 0.04 vs. 0.83 ± 0.03; 1.03 ± 0.06 vs. 1.01 ± 0.06) and absolute running speeds (15.00 ± 1.06 vs. 12.42 ± 1.22 km·h⁻¹; 19.04 ± 1.06 vs. 16.32 ± 1.29 km·h⁻¹) at both thresholds (p < 0.05), whereas women showed higher muscle oxygenation in vastus lateralis (60.44 ± 21.21 vs. 26.38 ± 10.21%) and fractional utilization of maximal aerobic speed (93.64 ± 6.44 vs. 91.43 ± 3.21%) at VT₂ (p < 0.01). Also, rate of perceived exertion was similar between sexes at both thresholds. Conclusion: Males showed higher absolute physiological values, while females demonstrated greater fractional utilization at VT₂ and higher muscle oxygenation. No sex differences were observed in rate of perceived exertion. These findings highlight the importance of using ventilatory thresholds in training prescription.

1. Introduction

Exercise intensity constitutes a fundamental variable in the prescription of endurance training. The training intensity distribution reflects the percentage of training volume an athlete dedicates to distinct intensity domains.

The three-zone training model proposed by Skinner and McLellan [1] in endurance sports is commonly employed to quantify such distribution [2,3,4]. These zones are typically delineated through the following two key metabolic thresholds: the first and second lactate/ventilatory thresholds (LT₁/VT₁ and LT₂/VT₂, respectively) [5]. In this context, gas exchange analysis remains the gold standard to identify these thresholds, enabling the accurate determination of the VT₁ and VT₂ thresholds [6].

Traditionally, exercise intensity has been prescribed using a fixed percentage of maximum values (e.g., $\dot{\mathrm{V}}$O_2max or HR_max). However, this method fails to unify a physiological response across individuals, as it tends to widely differ between individuals in acute responses [7] and chronic adaptations [8]. For instance, $\dot{\mathrm{V}}$O_2max percentage at which critical power (CP)—a proxy for VT₂—is obtained may widely differ among individuals [9]. Even when exercising at the same %$\dot{\mathrm{V}}$O_2max, individuals whose workload represents a higher percentage of CP tend to exhibit better improvements in endurance performance. Thus, prescribing training intensity based on physiological thresholds (e.g., ventilatory thresholds) has been suggested to reduce interindividual variability in metabolic stress and exercise tolerance [9,10].

Sex-based differences in endurance running performance have been well documented. Males and females differ in musculoskeletal, cardiovascular, molecular, and metabolic characteristics [11]. These sex-based differences are produced by the sex chromosome complement, sex hormones, and the epigenome and transcriptome on a molecular level [12]. This performance gap has been reported to be 2.4% [13]. Male athletes exhibit higher values of fat-free mass, blood volume, and absolute and relative maximal oxygen uptake [14,15]. Another parameter that could help to explain these differences in endurance running performance is muscle oxygen saturation (SmO₂) measured by near-infrared spectroscopy (NIRS). NIRS is emerging as a potential valuable marker for monitoring training intensity. Breakpoints in SmO₂, particularly in the vastus lateralis, have been associated with ventilatory thresholds in running [16] and cycling [17]. Despite these findings, it is essential to consider that SmO₂ measurements may differ depending on the specific muscle and sex differences. For instance, during running, the gastrocnemius muscle exhibits lower SmO₂ values compared to the vastus lateralis [18]. Also, during cycling, men tend to experience a greater decrease in SmO₂ as power output increases [19]. These findings underscore the need to consider both the specific muscle being evaluated and the individual’s sex when interpreting SmO₂ data, since women tend to experience higher oxygen saturation, likely due to a greater vasodilatory response [20].

Concerning physiological thresholds, Iannetta, et al. [21] reported that LT₁ and the maximal lactate steady state (MLSS, used to distinguish between heavy and severe intensity domains) occur at higher %$\dot{\mathrm{V}}$O_2max and %HR_max in female athletes compared to male athletes with the same level of performance. This suggests that both aerobic and anaerobic thresholds may occur at different relative (%$\dot{\mathrm{V}}$O_2max) intensities depending on each sex [22]. Also, physiological markers such as HR or %HR_max also exhibit sex-based variability [22].

Conversely, subjective measures such as rating of perceived exertion (RPE) has been proposed as an alternative for intensity prescription [23] due to their strong correlation with physiological markers, including HR [24], ventilation, respiratory frequency [25,26], and relative $\dot{\mathrm{V}}$O₂ [27]. Garcin, et al. [28] reported no sex differences in RPE at both relative (%$\dot{\mathrm{V}}$O_2max and %HR_max) and absolute (HR) intensities when using the Borg 6–20 scale. However, other studies have reported higher RPEs in women at equivalent absolute $\dot{\mathrm{V}}$O₂ values. Although these differences disappear when data were normalized to %$\dot{\mathrm{V}}$O_2max [29]. These conflicting results underscore the need for further investigation into sex-based differences in perceived exertion.

While sex-based physiological differences are well established, limited research has specifically examined how these differences manifest at ventilatory thresholds (VT₁ and VT₂) in trained endurance runners, particularly when integrating objective physiological markers (e.g., SmO₂) and subjective perceptual responses (RPEs). Furthermore, previous studies have often evaluated physiological thresholds or perceptual markers in isolation, without a comprehensive analysis across multiple physiological systems (respiratory, muscular, and perceptual) during GXT. By assessing cardiopulmonary parameters at ventilatory thresholds, muscle oxygenation in different muscles, and perceived exertion in male and female athletes, this study aims to provide a more integrative understanding of sex-specific endurance performance profiles. This may contribute to refining training prescription strategies using both objective and subjective parameters, attending sex-related differences. Therefore, this study aimed to compare trained male and female athletes regarding physiological, perceptual, and performance variables at VT₁ and VT₂.

2. Materials and Methods

2.1. Participants

An a priori sample size calculation for an independent-sample t-test was conducted based on effect sizes (ESs) reported in previous research within the field by Mendonca, et al. [30]. The estimated ESs for sex differences in VO₂ (ml·kg⁻¹·min⁻¹) at VT₁ and VT₂ indicated that a minimum of 14 and 7 participants per group, respectively, would be required to detect significant differences. In contrast, the ESs related to fractional utilization at VT₁ and VT₂ suggested that at least 12 and 19 participants per group, respectively, would be necessary to achieve adequate statistical power. These estimations were tested with a two-tailed independent-sample t-test (1 − β = 0.9; α = 0.05) using G*Power (v3.1) software. A total of 43 graded exercise tests (GXTs) from trained runners were analyzed, including 24 males and 19 females. Participants were selected using convenience sampling, and the recruitment process was carried out by contacting local running clubs (Figure 1). Athletes included in the study met the following inclusion criteria: running at least three days per week without injuries during the previous three months prior to the study and 10 km race performances faster than 35 min for males and 45 min for females. Exclusion criteria included running fewer than three days per week, having sustained any injury during the three months prior to the study, or not meeting the 10 km performance times. The descriptive characteristics of the participants are presented in

Table 1. Descriptive characteristics of the participants.

Variables	Males (n = 24)	Females (n = 19)
Age (y)	27.92 ± 6.43	24.37 ± 4.41
Body mass (kg)	61.75 ± 4.32	52.64 ± 4.11
Height (cm)	174.57 ± 5.80	165.00 ± 5.00
WAs in 10 km (points)	835.67 ± 128.36	816.68 ± 202.70
Maximal aerobic speed (km·h−1)	20.83 ± 0.87	17.42 ± 1.31
Maximal heart rate (bpm)	186.63 ± 8.20	189.05 ± 11.49

Note: Data are shown as the mean ± SD. WAs, World Athletics score.

.

Before the study, all participants were informed about the testing protocols and the possible risks; moreover, they provided written informed consent. The study was performed according to the principles of the Declaration of Helsinki (December 2013, Brazil), and the experimental protocols were approved by the ethics committee of the local university (CEIC926).

2.2. Study Design

This study followed a cross-sectional comparative design. All data collection was performed in a laboratory under similar environmental conditions for each participant (529 m altitude, 20–25 °C, and 35–40% relative humidity) without exhaustive exercise 48 h before the test. Participants were instructed to avoid caffeine and alcohol intake 24 h before each visit. All participants performed the test without advanced footwear technology, specifically, shoe models featuring a curved carbon fiber plate in the midsole to increase the longitudinal bending stiffness, a thicker and more compliant resilient midsole foam, and a more rocker shape than traditional shoe models [31].

2.3. Measurements

First, height was measured to the nearest 0.1 cm, and body mass was assessed to the nearest 0.1 kg with a portable stadiometer and caliber (Seca, Bonn, Germany). Then, the participants completed a GXT to exhaustion. The test started with 5 min of warm-up at a speed of 8 km·h⁻¹ for females and 10 km·h⁻¹ for males on a treadmill (HP Cosmos Pulsar, Nussdorf-Traunstein, Germany). Then, the starting speed of the GXT was 10 km·h⁻¹ for females and 12 km·h⁻¹ for males. The speed increased continuously by 1 km·h⁻¹ every minute until volitional exhaustion, while the incline was maintained at 1% [32], resulting in a duration of 8–12 min. Oxygen uptake ($\dot{\mathrm{V}}$O₂) and carbon dioxide output ($\dot{\mathrm{V}}$CO₂) were measured using a breath-by-breath gas analyzer (CPX/D Med Graphics, St. Paul, MN, USA). The analyzer was calibrated before each test with a known gas mixture (12% O₂ and 5% CO₂), and the volume sensor was calibrated with a 3 L syringe.

During the GXT, muscle oxygen saturation was measured at the right vastus lateralis (at ⅓ the distance from the patella to greater trochanter with the participant seated with knees bent to 90°) and gastrocnemius (external gastrocnemius, on the area with the largest diameter of the leg) using a wearable NIRS sensor (Moxy Monitor, Fortiori Designs, LCC., Hutchinson, MN, USA). The Moxy monitor is a wearable continuous wave NIRS sensor that can measure scaled heme volume, which represents both hemoglobin and myoglobin concentrations in the tissue, as well as SmO₂, on a 0–100% scale [33]. The sensor was secured with the manufacturer-supplied light shield and with adhesive tape to minimize signal interference from ambient light and movement. Moxy employs four wavelengths of near-infrared light (680, 720, 760, and 800 nm), with a single LED source and two detectors at 12.5 and 25 mm separations, providing a maximum penetration depth of approximately 12.5 mm [34]. It is recommended that the participant’s skinfold thickness (SF) be less than the maximum penetration depth (SF < ½ the maximum inter-optode distance); however, to better represent real-world use of NIRS with competitive athletes, no exclusion was made based on skinfold measurements. The SmO₂ was recorded at 0.5 Hz and smoothed with a 5 s symmetrical moving average to 1 Hz, as per manufacturer-recommended settings.

Likewise, absolute (W) and relative (W·kg⁻¹) running power were measured at each step of the GXT using an inertial measurement unit (Stryd Power Meter, Stryd Inc., Boulder, CO, USA), with a sampling frequency of 1000 Hz. Heart rate was recorded using a Polar H10 (Polar Electro Oy, Kempele, Finland). Finally, the rating of perceived exertion (RPE, scale 1–10) was asked at every stage. All participants had previous experience using the RPE 1–10 during the GXT at training sessions and received standardized instructions on how to use the RPE 1–10 scale before the test.

Following the GXT, first and second ventilatory thresholds were determined using a mixed-method approach (see Figure 1 and Figure 2 in Keir, et al. [35]). Two experienced researchers (S.R.-B. and F.G.-M.) individually checked the VT₁ and VT₂ values, and any discrepancies were resolved by consensus. Subsequently, the following physiological and performance variables were analyzed at each threshold: absolute speed, fractional utilization of speed relative to maximal aerobic speed (MAS), absolute HR and %HR_max, respiratory exchange ratio (RER), $\dot{\mathrm{V}}$O₂, energy cost (kJ·kg⁻¹·km⁻¹), and muscle oxygen saturation (SmO₂). Due to the short stage duration (1 min) and in line with previous recommendations [36], the last 30 s of each stage were used for analysis to minimize the influence of mean response time in physiological parameters, such as $\dot{\mathrm{V}}$O₂, HR, and SmO₂, and to allow participants to reach steady state. The average oxygen uptake during the last 30 s in the final stage of the GXT was considered as $\dot{\mathrm{V}}$O_2max when at least two of the following criteria were fulfilled [37]: (1) a plateau in $\dot{\mathrm{V}}$O₂ (an increase of less than 1.5 mL·kg⁻¹·min⁻¹ in two consecutive workloads); (2) RER > 1.15; (3) maximal HR values above 95% of the age-predicted maximum (220-age). The minimal necessary speed to reach $\dot{\mathrm{V}}$O_2max was considered MAS [38]. HR_max was considered the highest 5 s average recorded during the GXT [39].

2.4. Statistical Analysis

Data are represented as the mean ± SD. Data were screened for normality using a Shapiro–Wilk test. Sex differences were evaluated with an unpaired-samples t-test. Additionally, Cohen’s d effect size (ES) was also used for a better interpretation of the results. Effect sizes were considered trivial (<0.2), small (<0.6), moderate (<1.2), large (<2), or very large (>2) [40]. Analysis was performed with JASP (version 0.13.1 for Mac OS, JASP Team, Amsterdam, The Netherlands), and the level of significance used was p < 0.05. Figures were created with GraphPad Prism software (version 9.3.1 for Windows, Boston, United States).

3. Results

Table 2. Physiological and perceptual variables at first and second ventilatory thresholds in both sexes.

	First Ventilatory Threshold				Second Ventilatory Threshold
	Males	Females	p-Value	ES	Males	Females	p-Value	ES
Absolute power (W)	267.50 ± 23.19	194.63 ± 18.86	<0.001	3.41	333.96 ± 28.66	248.63 ± 24.03	<0.001	3.20
Relative power (W)	4.34 ± 0.35	3.71 ± 0.36	<0.001	1.77	5.42 ± 0.41	4.72 ± 0.37	<0.001	1.77
Running power (%MAS)	72.22 ± 4.20	73.63 ± 4.78	0.156	−0.31	90.44 ± 4.53	93.93 ± 3.91	0.006	−0.82
Speed (km·h−1)	15.00 ± 1.06	12.42 ± 1.22	<0.001	2.27	19.04 ± 1.06	16.32 ± 1.29	<0.001	2.32
Fractional utilization (%)	72.04 ± 4.91	71.36 ± 5.34	0.334	0.13	91.43 ± 3.21	93.64 ± 6.44	0.004	−0.83
Absolute HR (bpm)	160.38 ± 9.97	166.61 ± 13.84	0.049	−0.53	181.52 ± 8.10	184.61 ± 10.80	0.151	−0.33
%HRmax (bpm)	85.95 ± 4.10	88.28 ± 3.48	0.030	−0.61	97.33 ± 1.44	97.93 ± 1.13	0.078	−0.46
RER	0.87 ± 0.04	0.83 ± 0.03	0.001	0.97	1.03 ± 0.06	1.01 ± 0.06	0.032	0.59
VO2 (mL·kg−1·min−1)	51.15 ± 4.67	44.23 ± 5.64	<0.001	1.35	62.83 ± 5.80	53.60 ± 6.17	<0.001	1.55
VO2 (mL·kg−0·75·min−1)	67.80 ± 5.94	58.80 ± 7.47	<0.001	1.35	83.29 ± 7.35	71.26 ± 8.19	<0.001	1.55
SmO2 gastrocnemius (%)	41.21 ± 18.36	55.11 ± 17.77	0.008	−0.77	29.03 ± 17.73	37.31 ± 17.41	0.066	−0.47
SmO2 vastus lateralis (%)	44.99 ± 10.45	77.56 ± 12.16	<0.001	−2.89	26.38 ± 10.21	60.44 ± 21.21	<0.001	−2.11
Δ SmO2 gastrocnemius (%)	31.38 ± 20.65	20.64 ± 15.81	0.035	0.57	51.98 ± 23.05	47.32 ± 18.10	0.238	0.22
Δ SmO2 vastus lateralis (%)	20.03 ± 11.92	5.22 ± 8.65	<0.001	1.40	53.27 ± 17.23	26.57 ± 22.11	<0.001	1.36
RPE (1–10 scale)	3.65 ± 1.46	3.21 ± 1.47	0.169	0.30	8.00 ± 1.45	7.84 ± 1.30	0.359	0.11

Note: Data are shown as the mean ± SD. Comparisons were made with the student t-test. RER, respiratory exchange ratio; HR, heart rate; %HRmax, percentage of maximum heart rate; RPE, rating of perceived exertion; Δ, difference between rest values and threshold values; MAS, maximal aerobic speed; ES, effect size (Cohen’s d).

presents the results of physiological and perceptual variables at VT₁ and VT₂ in males and females.

3.1. Differences at VT₁

At VT₁, significant differences were observed between sexes in several parameters. Absolute running power at VT₁ was significantly (p < 0.001) greater in males (267.50 ± 23.19 W) than in females (194.63 ± 18.86 W). Likewise, relative running power (normalized to body weight) was higher in males (4.34 ± 0.35 W·kg⁻¹) compared to females (3.71 ± 0.36 W·kg⁻¹; p < 0.001). However, there was no significant difference in running power relative to MAS (p >0.05).

Moreover, males exhibited significantly higher (p < 0.001) speed at VT₁ (15.00 ± 1.06 km·h⁻¹) compared to females (12.42 ± 1.22 km·h⁻¹). There were significant differences (p < 0.05) in absolute HR and %HR_max between sexes, with higher absolute values and percentages in females (166.61 ± 13.84 bpm and 88.03 ± 3.42%) than in males (160.38 ± 9.97 bpm and 85.95 ± 4.10%).

RER was also significantly (p = 0.001) different between sexes, showing males a higher value (0.87 ± 0.04) than females (0.83 ± 0.03). Similarly, $\dot{\mathrm{V}}$O₂ at VT₁ was significantly higher (p < 0.001) in males (51.15 ± 4.67 mL·kg⁻¹·min⁻¹) than in females (44.23 ± 5.64 mL·kg⁻¹·min⁻¹). When body mass was adjusted to the power of 0.75, $\dot{\mathrm{V}}$O₂ at VT₁ remained higher in males (67.80 ± 5.94 mL·kg^−0.75·min⁻¹) than in females (58.80 ± 7.47 kJ·kg^−0.75·km⁻¹; p < 0.001).

Finally, regarding SmO₂ (Figure 2), females exhibited significant (p < 0.01) higher values than males in both the gastrocnemius (55.11 ± 17.77 vs. 41.21 ± 18.36%) and vastus lateralis (77.56 ± 12.16 vs. 44.99 ± 10.45%; p < 0.001). On the other hand, RPEs did not differ significantly between males (3.65 ± 1.46) and females (3.21 ± 1.47; p = 0.169).

3.2. Differences at VT₂

At VT₂, significant differences were observed between sexes in several parameters.

Regarding running power, males demonstrated higher (p < 0.001) absolute running power (333.96 ± 28.66 W) compared to females (248.63 ± 24.03 W). Similarly, running power relative to body weight was greater (p < 0.001) in males (5.42 ± 0.41 W·kg⁻¹) than in females (4.72 ± 0.37 W·kg⁻¹). However, running power relative to MAS was lower (p < 0.01) in males (90.44 ± 4.53%) than in females (93.93 ± 3.91%).

Concerning speed, males showed a meaningful higher (p < 0.001) speed at VT₂ (19.04 ± 1.15 km·h⁻¹) compared to females (16.32 ± 1.29 km·h⁻¹). In contrast, no significant differences were found in absolute HR and %HRmax, with males averaging (181.52 ± 8.10 bpm and 97.33 ± 1.44%; p = 0.151), and females (184.61 ± 10.80 bpm and 97.93 ± 1.13%; p > 0.05).

RER was higher (p < 0.05) in males (1.03 ± 0.06) than in females (1.01 ± 0.06). Similarly, $\dot{\mathrm{V}}$O₂ was greater (p < 0.001) in males (62.83 ± 5.80 mL·kg⁻¹·min⁻¹) than in females (53.60 ± 6.17 mL·kg⁻¹·min⁻¹). When adjusted body mass to the power of 0.75, $\dot{\mathrm{V}}$O₂ remained higher in males (83.29 ± 7.35 mL·kg^−0.75·min⁻¹) than in females (71.26 ± 8.19 mL·kg^−0.75·min⁻¹; p < 0.001). Females exhibited a higher (p < 0.01) fractional utilization of MAS at VT₂ (Figure 3) (93.64 ± 6.44%) compared to males (91.43 ± 3.21%).

In terms of SmO₂ (Figure 2), females had higher (p < 0.001) SmO₂ in the vastus lateralis (60.44 ± 21.21%) compared to males (26.38 ± 10.21%), while no significant differences were observed in SmO₂ in the gastrocnemius (males: 29.03 ± 17.73%; females: 37.31 ± 17.41%; p = 0.066). Finally, RPEs remained similar between groups (males: 8.00 ± 1.45; females: 7.84 ± 1.30; p = 0.359).

4. Discussion

This study aimed to define sex-based differences in physiological, perceptual, and performance variables at ventilatory thresholds (VT₁ and VT₂) in trained endurance runners. While sex-related physiological differences are well established, previous research has typically analyzed these factors separately. The main findings are that at both ventilatory thresholds, males had significantly higher absolute speed, $\dot{\mathrm{V}}$O₂, RER, and absolute and relative power outputs than females. Females showed greater SmO₂, especially in the vastus lateralis, and slightly higher fractional utilization of MAS at VT₂ but not at VT₁. Heart rate was different at VT₁ but similar at VT₂. Also, rate of perceived exertion was similar between sexes at both thresholds. According to McClelland and Weyand [13], performance differences between males and females have been observed across different-duration running events. In regard to these sex-related physiological differences, they are not merely descriptive, as they have practical implications for training prescription. Understanding these differences is crucial to prescribe endurance training programs correctly, set appropriate intensity zones (e.g., based on ventilatory thresholds), and avoid over- or under-prescription of training loads. Our findings contribute to the literature by providing a more integrative perspective on how these differences manifest across systems during graded exercise. This may help enable more precise training prescription strategies by considering both absolute and relative internal and external load responses specific to each sex.

4.1. Absolute Speed, Fractional Utilization of MAS, and Running Power

Sex-based differences were found at VT₁ and VT₂ speeds. On average, males exhibited 18.82% (15.00 ± 1.06 vs. 12.42 ± 1.22 km·h⁻¹) and 15.39% (19.04 ± 1.06 vs. 16.32 ± 1.29 km·h⁻¹) higher speeds at VT₁ and VT₂, respectively. On the other hand, when analyzing speed as fractional utilization of MAS, the only differences were found at VT₂; females exhibited 2.38% (91.43 ± 3.21 vs. 93.64 ± 6.44) higher fractional utilization of MAS. This phenomenon was previously observed by Mendonca, Matos and Correia [30], who found that in recreational runners, females exhibited higher fractional utilization at both ventilatory thresholds (p < 0.05), lower MAS (p < 0.001), and less absolute speed at ventilatory thresholds (p < 0.05). In cycling, similar results may be observed, despite males having a higher power output at both ventilatory thresholds (p < 0.001), females exhibited higher power output relative to peak power at both ventilatory thresholds (p < 0.001) [41]. The higher fractional utilization of MAS observed in females at VT₂ suggests that men and women (of a similar endurance performance level), may perform at a different percentage of $\dot{\mathrm{V}}$O_2max during submaximal efforts. These results underscore that women can perform at VT₂ speeds closer to their maximal aerobic capacity.

From a practical application, these data highlight the necessity of considering sex-specific physiological responses when designing training programs. Although males achieve higher absolute intensities (e.g., speed, $\dot{\mathrm{V}}$O₂, and running power) at both ventilatory thresholds, females may exhibit distinct relative adaptations, such as a higher fractional utilization of MAS at VT₂. This suggests that when prescribing endurance training based on relative intensity (e.g., %MAS), applying the same percentage thresholds to both sexes may not be the same physiological stimuli. An alternative method to prescribing training intensity is based on percentages of VT₁ and VT₂ thresholds, as they reduce exercise tolerance and acute metabolic responses compared to traditional intensity approaches (%$\dot{\mathrm{V}}$O_2max/%MAS) [9].

Moreover, given the close relationship between running power and speed (r = 0.98) [42], the observed differences in running power–both absolute and relative to body mass reflect sex-based differences in running mechanics and metabolic cost. When running power was normalized to MAS, differences only emerged at VT₂, suggesting that higher-intensity efforts may require greater individualization based on sex. To ensure that training targets the intended physiological zone, it is necessary to individualize according to the sex of the athlete. Therefore, these findings support the need to adjust endurance training prescriptions (e.g., interval intensities, threshold training sessions) considering both ventilatory thresholds (specifically VT₂).

4.2. Ventilatory and Muscle Oxygenation Parameters

When analyzing $\dot{\mathrm{V}}$O₂ expressed relative to body weight at both ventilatory thresholds, males had 14.15% (51.15 ± 4.67 vs. 44.23 ± 5.64 mL·kg⁻¹·min⁻¹, for males and females respectively) and 15.85% (62.83 ± 5.80 vs. 53.60 ± 6.17 mL·kg⁻¹·min⁻¹) higher $\dot{\mathrm{V}}$O₂ at VT₁ and VT₂, respectively. If expressed on an allometric scale, males had 14.22% (67.80 ± 5.94 vs. 58.80 ± 7.47 mL·kg^−0.75·min⁻¹) and 15.57% (83.29 ± 7.35 vs. 71.26 ± 8.19 mL·kg^−0.75·min⁻¹) higher $\dot{\mathrm{V}}$O₂ at VT₁ and VT₂, respectively. These results are similar to the previous ones found by Mendonca, Matos and Correia [30]. Another finding was that females exhibited a 4.71% lower RER at VT₁ (0.87 ± 0.04 vs. 0.83 ± 0.03) and 1.96% at VT₂ (1.03 ± 0.06 vs. 1.01 ± 0.06) than males. This aligns with previous studies showing that females rely more on fat oxidation and less on carbohydrate and protein sources during endurance exercise [43,44]. A potential explanation to this phenomenon, relates to differences in skeletal muscle fiber composition, as type 1 fibers–more prevalent in females—are more oxidative and rely on aerobic metabolism, while type II fibers—more dominant in males—favor glycolytic, carbohydrate-dependent pathways [45,46]. A previous study [47] reported no sex differences in RER during exercise at 70% and 90% of LT (analogous to VT₁ in our study), while others [48,49] demonstrated a lower RER in females during exercise prescribed at 90–100% of CP/MLSS (analogous to VT₂ in our study). In addition, exercise prescribed at Δ50 (intensity between LT and CP) eliminated sex differences in exercise substrate utilization [50].

These contradictory results underscore the necessity for additional studies about the impact of training prescription at ventilatory/lactate thresholds compared to prescription relative to %$\dot{\mathrm{V}}$O_2max/MAS.

Similarly, when analyzing SmO₂ values, males exhibited significantly lower levels in both the gastrocnemius and vastus lateralis muscles at VT₁ and specifically in the vastus lateralis at VT₂. Although a similar trend was observed in the gastrocnemius at VT₂, it did not reach statistical significance. These findings are in line with a previous study [19] that reported higher SmO₂ values in females across different muscle groups at the first and second lactate thresholds. In our study, females also showed significantly higher baseline SmO₂ in the gastrocnemius (68.26 ± 12.13%) and vastus lateralis (81.82 ± 10.27%) compared to males (58.91 ± 12.42% and 56.21 ± 10.50%, respectively) during the GXT (p < 0.05).

Furthermore, males experienced a greater decrease in SmO₂ in the vastus lateralis at both ventilatory thresholds but not in the gastrocnemius muscle. This finding is similar to those found by Espinosa-Ramírez, et al. [51] and may be related to sex differences in muscle fiber composition, contraction velocity, and cross-sectional area in locomotor muscles, which could result in increased oxygen demand and extraction during high-intensity exercise [52]. The present study assessed RER together with SmO₂ in two distinct muscles (vastus lateralis and gastrocnemius), providing complementary insights into both whole-body substrate utilization and local muscle oxygenation dynamics during exercise. While previous studies have described sex differences in substrate utilization or muscle oxygenation separately, this combined approach allows for a more integrated understanding of systemic and muscular responses, particularly around ventilatory thresholds.

These aspects could have relevant implications in training prescription and adaptations. For example, female athletes may sustain longer effort durations with higher oxygen supply, and the use of NIRS technology during training sessions could monitor internal load, adjusting the recovery period during high-intensity interval training. This integrative analysis offers a more complete picture of endurance physiology and highlights the potential of NIRS technology for individualized, real-time training load monitoring.

4.3. Heart Rate and Rate of Perceived Exertion

Despite the physiological differences observed in absolute performance variables, the comparable heart rate responses suggest a similar level of cardiovascular strain at VT₂ for both sexes. This phenomenon has been previously reported in other sports like ski mountaineering, where males and females did not exhibit significant differences in absolute heart rate at both ventilatory thresholds [53]. However, there were significant differences in absolute heart rate and %HR_max at VT₁. Female athletes exhibited 2.67% (85.95 ± 4.10 vs. 88.28 ± 3.48%) and 3.81% (160.38 ± 9.97 vs. 166.61 ± 13.84 bpm) higher %HR_max and absolute heart rate, respectively. When comparing HR_max, no significant differences were found between sexes (186.63 ± 8.20 vs. 189.05 ± 11.49). These results suggest that females, as observed with the fractional speed of MAS for the second ventilatory threshold, can operate closer to their maximal capacities at the first ventilatory threshold for cardiovascular variables.

Although the laboratory-defined intensity thresholds can be established as the gold-standard method, it is less accessible to all athletes, especially those of a lower level. In our study, there were no significant differences between sexes in RPE (scale 1–10) at both ventilatory thresholds (~3 and ~8 for VT₁ and VT₂, respectively). These findings align with those found by Coe and Astorino [54], who found no sex differences with an RPE scale of 6–20. These findings align with those found by Coe and Astorino [54], who found no sex differences with an RPE scale of 6–20. In addition, RPE values (scale 6–20) at LT₁ (~12) and LT₂ (~15) were similar between females and males in a study by Nuuttila, et al. [55]. Therefore, this alignment among internal load markers supports their reliability as tools for prescribing training intensity across sexes and reinforces the potential of using these parameters for individualized training prescription without the need for sex-specific adjustments in these metrics.

4.4. Limitations

One limitation of our study is the lack of measurement of the adipose tissue thickness of the vastus lateralis and gastrocnemius muscles, as female participants tend to have greater thickness. This could reduce the accuracy of muscle oxygenation estimates by altering the signal captured, reflecting subcutaneous tissue rather than muscle. However, we consider that the participants in this study (well-trained runners) represent a specific athletic population in which adipose tissue thickness is generally low, regardless of sex. Furthermore, Ref. [56] reported that even without exclusion or correction for subcutaneous fat, the Moxy device offers good reliability.

4.5. Practical Applications

The study emphasizes important sex-related variations in physiological responses that call for customized training strategies. While males typically demonstrate higher absolute values in measures such as running speed, power output, and oxygen consumption at ventilatory thresholds, females exhibit greater fractional utilization of MAS and higher SmO₂ during submaximal efforts. This suggests trained female athletes operate closer to their maximal capacities at comparable relative intensities. For example, females reached VT₂ at 93% of MAS compared to 91% in males, despite both sexes achieving similar relative intensities at VT₁ (~72% of MAS). These differences imply that prescribing training intensities based on fixed percentages of MAS may lead to errors. A male athlete with a MAS of 22 km·h⁻¹ and VT₂ at 20 km·h⁻¹ exercising at 93% MAS (20.5 km·h⁻¹) would enter the severe-intensity domain, whereas a female athlete with a MAS of 19 km·h⁻¹ and VT₂ at 18 km·h⁻¹ at the same percentage (17.7 km·h⁻¹) would remain in the heavy domain. To address this, coaches should prioritize threshold-based intensity zones over generic percentage targets, ensuring training aligns with individual physiological profiles and minimizes sex-related discrepancies. Therefore, RPE can be effectively used to guide and monitor training intensity without requiring sex-specific adjustments, facilitating their integration into daily training environments.

5. Conclusions

The findings of this study confirm that there are notable physiological differences between trained males and females at both ventilatory thresholds. In absolute terms, males exhibited higher values in parameters such as running speed, oxygen consumption, and power output, which is consistent with known sex-related physiological advantages. However, the following three key observations are shown: (1) females reached VT₂ at a higher fractional utilization of MAS compared to males; (2) muscle saturation was higher in women at both ventilatory thresholds in vastus lateralis and gastrocnemius; and (3) no significant differences were found for RPEs at both ventilatory thresholds between the sexes. These findings may indicate that, despite having lower absolute capacities, trained females could operate relatively more efficiently within their physiological limits, potentially maintaining submaximal intensities closer to their maximal capacity. These results highlight the importance of interpreting endurance performance not only through absolute metrics but also through the lens of relative physiological capacity. These findings reinforce the importance of using relative physiological metrics—specifically, those based on ventilatory thresholds such as VT₁ and VT₂—alongside absolute values to better interpret endurance performance and guide training prescriptions.