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Based on a minimum increase in C of 5% (~0.3 J kg-1 m-1), a coefficient of variation of 5%, an alpha error of 0.05 and a power of 90%, the minimal number of athletes required for the group was 11. Twelve healthy men, who were national level athletes of AR (age 31.3 ± 7.7 years, height 1.81 ± 0.05 m, body mass 75.5 ± 9.1 kg, training volume 39.12 ± 9.02 km per week, AR experience 64 ± 49 months), carried out three maximal progressive (VO2max protocol) and three submaximal constant-load (running cost protocol) tests, defined in the following quasi-randomized conditions: unloaded, 7% and, 15% of body mass. We choose percent loads due to inherent effects of absolute load on performance. Also, we settle these percent loads because are the loads usually used in AR [11]. All participants gave their written informed consent to participate in the study. All procedures followed were in accordance with the ethical standards and with the Helsinki Declaration of 1975, as revised in 2008, and were approved by the responsible local Ethics Committee of the Universidade Federal do Rio Grande do Sul on human experimentation.
During the preliminary visit, athletes were familiarized with all loads, equipment, and protocols. All tests were separated by about 2–4 days. Firstly, the three maximal running tests at 0, 7% and 15% of individuals’ body mass were randomized in three visits and, in the fourth visit, the submaximal tests were again randomized. The athletes used their backpacks to perform the bouts with the extra load. The backpack position was set between the first thoracic and lumbar vertebra, and it was fixed to avoid excessive oscillation (Fig 1). In all tests, the heart rate (Polar, Kempele, Finland), end-tidal partial pressure of oxygen, end-tidal partial pressure of carbon dioxide, oxygen uptake, carbon dioxide output and ventilation per minute (MEDGRAPHICS, CPX/D, Diagnostic Systems, Saint Paul, Minnesota, USA) were measured continuously. The gas data were registered breath-by-breath. Temperature, atmospheric pressure and humidity in the laboratory were 20 ± 2°C, 1026 ± 10 mmHg, and 50 ± 8%, respectively. 6–20 Borg’s ratings of perceived exertion scale (RPE) was shown to the athletes during the last 30 s of each stage (maximal tests) and just after the end of submaximal tests. Each athlete received detailed instructions about the use of the scale before the beginning of the first test. The total time at each maximal test was 30 minutes, and 1 hour to the submaximal protocol.
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0189516.g001 The athlete and his backpack with the extra load. Before the trials, the athletes performed a warm-up walking on a treadmill (QUINTON, ST55, New York, USA) inclined at 1% for five minutes at 6.0 km h-1 [12]. During the warm-up before the tests with load, the backpack with the respective load (7 or 15% of body mass) was adjusted. They were familiarized to treadmill exercise. The initial speed of the maximal tests was 6.0 km h-1, and the increment was 1.0 km h-1 per minute until subjects reached volitional exhaustion.
Initially, resting oxygen uptake was measured in orthostasis, during 5 min. The individuals were asked to carry-out a warm-up for five minutes walking on a treadmill. Taking into account the individual outcomes from maximal tests, the athletes carried out the submaximal tests at the intensity associated with 10% below the second ventilatory threshold according to the respective condition (0%, 7%, and 15% of the individual’s body mass), all on the same day. The duration of each test was 6 min. On average, 10 minutes of rest between submaximal tests were enough to achieve the initial heart rate and oxygen consumption. The rates of oxygen consumption and carbon dioxide production were measured continuously during each trial. The heart rate in all submaximal tests was not greater than 80 percent of the maximal heart rate. Besides, the respiratory exchange ratio was also monitored achieving values lower than one.
The VO2max, the velocity associated with VO2max (vVO2max), maximal RER, maximal heart rate, first and second ventilatory thresholds, and velocity associated with first and second ventilatory thresholds (v1Tvent and v2Tvent, respectively) were determined using computerized indirect calorimetry system [13]. The highest average of five oxygen consumption values was interpreted as the VO2max [14]. The value was considered valid when, at least, one of following criteria was observed: i) estimated maximal heart rate; ii) plateau on oxygen consumption with concomitant increase in the speed (all subjects attained the true VO2max); iii) respiratory exchange ratio greater than 1.1; iv) rating of perceived exertion greater than 17 (very hard) relative to Borg scale. The first and second ventilatory thresholds were determined according to the method proposed by [15]. The first ventilatory threshold (also denominated as individual ventilatory threshold) was determined from the first increase in ventilation-minute with a rapid rise in the ventilatory equivalent of oxygen consumption with no concomitant increase in the ventilatory equivalent of carbon dioxide production curve. The second ventilatory threshold (also denominated as respiratory compensation point) was defined as follows: i) a systematic increase in the ventilatory equivalent of oxygen consumption; ii) a concomitant nonlinear increase in the ventilatory equivalent of carbon dioxide production; and iii) a reduction in the difference in the inspired and end-tidal oxygen pressure. The ventilatory thresholds were determined in a blinded way by two independent evaluators.
The submaximal oxygen uptake and heart rate were averaged from the last 60 s of the test [16]. The running economy was denoted by C, expressed in J kg-1 m-1. For that, we divided the net metabolic rate (gross—stand metabolic rate) by speed and we converted oxygen in ml to Joules relative to combustion enthalpy of substrates resulting from oxidation observed indirectly from respiratory exchange ratio [17]. The metabolic rate (ECO) was also calculated and expressed in ml kg-1 min-1. The maximal and submaximal metabolic power values were normalized to body mass and expressed in ml kg-1 min-1. Recently, we showed that the relationship between metabolic parameters and performance is independent of how the parameters are relativized in runners [18–20]. All data can be seen in the supplementary material (S1 Table).
The Shapiro-Wilk test was used to verify data normality. We performed the descriptive statistics calculating mean ± standard deviation. The Pearson product-moment correlation test was carried out in order to test the relationship among the physiological determinants of performance (VO2max, C and, ventilatory threshold) with different load conditions. The linear regression analysis was used to estimate the speeds associated empirically with ventilatory thresholds when carrying loads. Possible differences between conditions (0, 7 and 15% of body mass) were analyzed using the repeated-measures analysis of variance (ANOVA) with Bonferroni post hoc test. To verify the possibility of violation of the assumption of sphericity, we applied the Mauchly test using the Greenhouse-Geisser correction for all analyses. Significance was accepted at P ≤ 0.05, statistical power was 90%, and the analyses were performed in Statistical Package for Social Sciences version 20.0 (SPSS, Chicago, Illinois, USA). We used the Cohen’s d coefficient to determine the effect sizes [21]. We determined the differences in proportions using the rule of thumb criteria set out by Hopkins: trivial (< 0.2), small (0.2–0.6), moderate (0.6–1.2), or large (> 1.2).
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