RESEARCH ARTICLE Oxygen uptake kinetics and energy system’s contribution around maximal lactate steady state swimming intensity Jailton Gregório Pelarigo1,2,3,4☯*, Leandro Machado3,4‡, Ricardo Jorge Fernandes3,4‡, Camila Coelho Greco5‡, João Paulo Vilas-Boas3,4☯ 1 University Catholic Center of Quixadá–UNICATÓLICA, Quixadá, Ceará, Brazil, 2 Metropolitan College of Grande Fortaleza–FAMETRO, Fortaleza, Ceará, Brazil, 3 Centre of Research, Education, Innovation and Intervention in Sport, Faculty of Sport, University of Porto, Porto, Portugal, 4 Porto Biomechanics Laboratory, LABIOMEP, University of Porto, Porto, Portugal, 5 Human Performance Laboratory, Physical Education Department, São Paulo State University, Rio Claro, São Paulo, Brazil ☯ These authors contributed equally to this work. ‡ These authors also contributed equally to this work. * jailtongp@hotmail.com Abstract The purpose of this study was to examine the oxygen uptake ( _VO2) kinetics and the energy systems’ contribution at 97.5, 100 and 102.5% of the maximal lactate steady state (MLSS) swimming intensity. Ten elite female swimmers performed three-to-five 30 min submaximal constant swimming bouts at imposed paces for the determination of the swimming velocity (v) at 100%MLSS based on a 7 x 200 m intermittent incremental protocol until voluntary exhaustion to find the v associated at the individual anaerobic threshold. _VO2 kinetics (cardi- odynamic, primary and slow component phases) and the aerobic and anaerobic energy con- tributions were assessed during the continuous exercises, which the former was studied for the beginning and second phase of exercise. Subjects showed similar time delay (TD) (mean = 11.5–14.3 s) and time constant (τp) (mean = 13.8–16.3 s) as a function of v, but reduced amplitude of the primary component for 97.5% (35.7 ± 7.3 mL.kg.min-1) compared to 100 and 102.5%MLSS (41.0 ± 7.0 and 41.3 ± 5.4 mL.kg.min-1, respectively), and τp decreased (mean = 9.6–10.8 s) during the second phase of exercise. Despite the slow com- ponent did not occur for all swimmers at all swim intensities, when observed it tended to increase as a function of v. Moreover, the total energy contribution was almost exclusively aerobic (98–99%) at 97.5, 100 and 102.5%MLSS. We suggest that well-trained endurance swimmers with a fast TD and τp values may be able to adjust faster the physiological requirements to minimize the amplitude of the slow component appearance, parameter associated with the fatigue delay and increase in exhaustion time during performance, how- ever, these fast adjustments were not able to control the progressive fatigue occurred slightly above MLSS, and most of swimmers reached exhaustion before 30min swam. PLOS ONE | DOI:10.1371/journal.pone.0167263 February 28, 2017 1 / 12 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPENACCESS Citation: Pelarigo JG, Machado L, Fernandes RJ, Greco CC, Vilas-Boas JP (2017) Oxygen uptake kinetics and energy system’s contribution around maximal lactate steady state swimming intensity. PLoS ONE 12(2): e0167263. doi:10.1371/journal. pone.0167263 Editor: Juan Sastre, Universitat de Valencia, SPAIN Received: February 16, 2016 Accepted: November 11, 2016 Published: February 28, 2017 Copyright: © 2017 Pelarigo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This investigation was supported by grants of the Capes Foundation, Ministry of Education of Brazil (BEX: 0536/10-5): JGP and project PTDC/DES/101224/2008 (FCOMP-01- 0124-FEDER-009577): JPVB RF. Competing interests: The authors have declared that no competing interests exist. http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0167263&domain=pdf&date_stamp=2017-02-28 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0167263&domain=pdf&date_stamp=2017-02-28 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0167263&domain=pdf&date_stamp=2017-02-28 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0167263&domain=pdf&date_stamp=2017-02-28 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0167263&domain=pdf&date_stamp=2017-02-28 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0167263&domain=pdf&date_stamp=2017-02-28 http://creativecommons.org/licenses/by/4.0/ Introduction An important aspect of aerobic endurance performance is the ability to sustain the highest per- centage of maximal oxygen uptake (% _VO2max) as long as possible. In this sense, coaches and swimmers have used the % _VO2max in different submaximal intensities to control, prescribe and improve sports training [1]. Additionally, scientists have shown that the _VO2 kinetics analysis may help to understand the physiological adjustments produced over time by the ath- letes in several sports, allowing them to maintain a high % _VO2max in a physiological steady- state during aerobic endurance performance [2–4]. Meanwhile, the scientific community has mainly described the _VO2 kinetics in three differ- ent intensity domains during continuous exercise. First, the moderate domain is described as the exercise intensities in which a state steady for _VO2 is achieved within 3 min of constant exercise [5]. Subsequently, the heavy domain is described as the exercise intensities in which _VO2 slow component should be evident, causing a delay on the achievement of the _VO2 steady-state during exercise [2]. Last, the severe domain is described as the exercise intensities in which _VO2 is elevated compared to rest values and continue to increase over time, leading to attain the _VO2max [6, 7]. Maximal lactate steady state (MLSS) is considered one of the main relevant parameters for prescription and improvement of aerobic endurance performance, once it has been assumed as the limit intensity at which, during prolonged and submaximal exercise, the metabolic energy is produced mainly by the aerobic metabolism of pyruvate and glycolysis [8, 9]. More- over, MLSS is identified as the maximal intensity that can be maintained over time without the lactate production exceeding removal more than 1 mmol.L-1, and considered gold-standard method for the evaluation of aerobic capacity [10–12]. Once maximal velocity where a steady-state is found represents a fundamental physiologi- cal border, subtle changes in this intensity could likely modify _VO2 kinetics response. For instance, when the exercise is performed at intensities slightly below MLSS, a physiological steady state is sustained for both blood lactate concentration [La-] and _VO2 as a function of time [6, 7, 13]. On the other hand, at intensities above the MLSS, a significant increase in [La-] and _VO2 is likely to be observed throughout time [3, 7, 8, 12], leading to fatigue and voluntary exhaustion [3, 4, 14]. Moreover, the swimming MLSS determination needs a short time of interruption for the blood collection during the 10th minute of exercise for the analysis of [La- ], and then, a resumption of exercise to complete the test. Thus, it seems to be fundamental to examine the behavior of _VO2 kinetics not only the beginning of exercise, but too after the resumption of exercise throughout exercise to better understanding of the entire process of the swimmer physiological response along the exercise. _VO2 kinetics has been studied in different sports over the last decades [2, 6, 15], and there are relevant number of researches based on [La-] and gas exchange at intensities related to MLSS [8, 13, 14]. However, no study has evaluated _VO2 kinetics at (and around) the MLSS intensity. Thus, our purpose was to examine _VO2 kinetics and the energy systems’ contribu- tion at 97.5, 100 and 102.5%MLSS in swimming. It was hypothesized that at 97.5%MLSS, _VO2 kinetics adjustments may not be so evident such as 100 and 102.5%MLSS. It was further hypothesized that even at the 100%MLSS intensity, swimmers may also have to adjust _VO2 kinetics during the exercise, once this intensity would lead to voluntary exhaustion over time. On the other hand, at the intensity of 102.5%MLSS, _VO2 kinetics may be compromised by fatigue, requiring faster time adjustments for time delay and time constant, and higher _VO2 amplitudes either for primary or slow components compared to lower exercise intensities. We Oxygen kinetics and aerobic endurance performance PLOS ONE | DOI:10.1371/journal.pone.0167263 February 28, 2017 2 / 12 further intended to assess _VO2 kinetics of the second phase of exercise, starting after the collec- tion of [La-] and resumption of exercise (from 10th min to the exercise end—final exercise), hypothesizing that these parameters could be faster than without previous exercise. Moreover, as MLSS may be maintained for long time period without continuous [La-] accumulation, as well as a submaximal exercise, the energy supply should be mainly supported through the aer- obic system for the swimming intensities of ± 2.5% around MLSS. Material and methods Ten elite female swimmers volunteered and gave written informed consent (or parent/guard- ian when subjects were under 18yrs) to participate in the present study, which was approved by the Ethics Committee of Faculty of Sport from the University of Porto and performed according to the Declaration of Helsinki. The swimmers were (mean ± SD) 17.6 ± 1.9 years of age, 1.70 ± 0.05 m height, 61.3 ± 5.8 kg body mass, 15.5 ± 2.9% body fat mass, and 54.9 ± 6.7 mL.kg.min-1 _VO2max, specialized in middle- and long-distance swimming events. The subjects had, at the least, seven years of experience as competitive swimmers and their mean perfor- mance over a 400m freestyle swim was 88.0 ± 3.4% of the short course word record. The test sessions were performed in a 25 m indoor swimming pool. Air humidity was main- tained nominally between 40–60%, and pool water temperature between 27–28˚C. Swimmers were advised to refrain from intense training at least 24 h before the experimental sessions. The tests were conducted within a seven day period, at the same time of the day (± 2 h), mini- mizing the circadian rhythm effects. Previously to the test sessions, swimmers performed a 1000 m warm-up at low/moderate intensity. The tests were performed in front crawl, with in- water starts and open turns, without relevant underwater glides. A 24 h interval was imposed between all tests. Initially, swimmers performed an intermittent incremental protocol until voluntary exhaustion to find the velocity (v) corresponding to the individual anaerobic threshold (IAnT). The distance covered in each step was 200 m, with v increases of 0.05 m.s-1 and 30 s rest intervals between each swim [16]. According to these authors, the predetermined v of the last step was defined as the currently best expected performance for the subjects’ 400 m front crawl, and then used to define all the v steps for the incremental test. The IAnT was assessed by the relationship between [La-] and v using a curve fitting method, and considered the inter- ception point between linear and exponential regressions to determine the accurate v where [La-] increased exponentially [16, 17]. Subsequently, each swimmer performed three-to-five 30 min submaximal constant swim- ming bouts at imposed paces to determine the highest v where a MLSS was achieved (100% MLSS). The first trial was performed at the v corresponding to IAnT; and, if a steady state or a decrease in [La-] was observed, further subsequent trials with 2.5% higher velocities were per- formed until no [La-] steady state could be maintained [14]. Following this study, if the first trial resulted in a clearly identifiable increase of the [La-], and/or could not be sustained due to exhaustion, further trials were conducted with reduced velocities. MLSS was defined as the [La-] that increased by no more than 1 mmol.l-1 between the 10th and 30th min of the test [9]. Earlobe capillary blood samples (5 μL) were collected: (a) at rest and in the first 30 s after each step of the incremental test, immediately after exhaustion, and at each 2 min of recovery (until the [La-] recovery peak was found); and (b) at rest, 10 and 30th min (or voluntary exhaustion) of each continuous bout (Lactate Pro, Arkray, Inc., Kyoto, Japan). The v was set and maintained using a visual underwater pacer (GBK-Pacer, GBK Electron- ics, Aveiro, Portugal), with lights located each 2.5 m apart by a light strip on the bottom of the pool. Swimmers followed the flashing lights to maintain the predetermined velocities. and Oxygen kinetics and aerobic endurance performance PLOS ONE | DOI:10.1371/journal.pone.0167263 February 28, 2017 3 / 12 were instructed to keep their heads above each visual signal. Exhaustion was defined when the swimmers remained 5 m behind the lights. _VO2 was measured by a telemetric portable gas analyzer (K4b2, Cosmed, Italy) in both tests, connected to the swimmer by a low hydrodynamic resistance respiratory snorkel and valve system (New AquaTrainer1, Cosmed, Italy). This system has been previously validated [18] and used in similar studies [15]. The device was calibrated for minute ventilation ( _VE) with a calibrated syringe (3 L) and the O2 and CO2 analyzers with standard calibration gases (16% O2 and 5% CO2) before each test. In all tests, _VO2 data were analyzed and errant breaths occurred by swallow water and/or saliva, sighs and coughs were excluded. Afterwards, _VO2 values were measured in mean ± 3 SD and outside values were removed. Subsequently, the breath-by-breath data were linearly interpolated to provide five-by-five s values, and smoothed using three breath averages [15, 19]. Heart rate (HR) was monitored and registered continu- ously by a HR monitor system (Polar Vantage NV, Polar electro Oy, Kempele, Finland) and transferred in real time, through a telemetric signal, to the K4b2 device. The HR values were also averaged every 5 s intervals. The average _VO2 values were analyzed by a nonlinear least squares algorithm to fit the data through MatLab 7.0 Software (MathWorks, Natick, MA). The mathematical model consisted of two (cardiodynamic and primary components) or three (cardiodynamic, primary and slow components) exponential models. An F-Test (p< 0.05) was used to evaluate whether the two or three exponentials models provided the best fit to each data set. _VO2 ðtÞ ¼ _VO2 baseline þ Ac ½1 � e� ðt=t c Þ� Phase I ðcardiodynamic componentÞ þ Ap ½1 � e� ðt� TD p Þ=t p� Phase II ðprimary componentÞ þ As ½1 � e� ðt� TD s Þ=t s� Phase III ðslow componentÞ where _VO2 (t) represents the absolute _VO2 at time, _VO2 baseline is the _VO2 in resting baseline period, Ac and τc are the amplitude and the time constant of the cardiodynamic component; Ap, TDp and τp are the amplitude, the time delay and the time constant of the primary compo- nent; As, TDs and τs are the amplitude, the time delay and the time constant of the slow com- ponent. The mean response time (MRT) was applied to represent the overall pulmonary _VO2 kinetics response, which was determined as the sum of TDp and τp [15]. The _VO2 kinetics was assessed during the beginning of exercise until the break (at the 10th min) of swim for collec- tion of [La-] (initial exercise), and the second phase of exercise, starting after the collection of [La-] and resumption of exercise (final exercise). The energy systems’ contribution has been assessed by the total energy expenditure ( _E). The _E was obtained by the addition of the aerobic energy expenditure calculated by the differ- ence between the exercise _VO2 ( _VO2exercise) and baseline _VO2 ( _VO2baseline) (mL.kg-1.min-1), and by the anaerobic energy expenditure that was calculated by the net [La-] values transformed into O2 equivalents using the constant value of 2.7 mLO2.kg-1.mM-1 [15, 20] during continu- ous exercises. Data are presented as mean and standard deviation (± SD). Normality and sphericity of data were checked with the Shapiro-Wilk’s W and Mauchley Sphericity tests. When the assumption of sphericity was not attained, Greenhouse-Geisser or the Huynh-Feld adjusted univariate tests for repeated measures were used. The partial Eta square (p 2) was used to mea- sure the effect size, defined as small, medium and large for values of 0.01, 0.06 and 0.14, respec- tively [21]. The comparisons of _VO2 kinetics (cardiodynamic and primary components) and energy systems’ contribution (aerobic and anaerobic energy expenditure) were performed Oxygen kinetics and aerobic endurance performance PLOS ONE | DOI:10.1371/journal.pone.0167263 February 28, 2017 4 / 12 using multivariate ANOVA and examined by the intensity and previous exercise effects. The v and [La-] values were performed using the univariate ANOVA. All analyses were conducted for repeated measures, complemented with the Bonferroni correction post-hoc test with a sig- nificance level of p< 0.05. Results All swimmers performed 30 min when swimming at 97.5 and 100%MLSS, but eight swimmers were not able to maintain the predetermined v during 30 min at 102.5%MLSS, reaching volun- tary exhaustion at 19.3 ± 4.9 min. The average v and % _VO2max values were different in between the three swim intensities, with 97.5%MLSS slowest and lowest, and 102.5%MLSS fastest and highest (F2,18 = 2560.200, p< 0.001, p 2 = 0.996; F2,18 = 15.538, p< 0.001, p 2 = 0.633, respec- tively) (Table 1). [La-] and HR values for the three swim intensities are also shown in Table 1 with a higher values at 102.5%MLSS compared to 97.5 and 100%MLSS for [La-] (F2,18 = 18.123, p< 0.001, p 2 = 0.668), and at 102.5%MLSS compared to 97.5%MLSS for HR (F2,18 = 7.222, p< 0.005, p 2 = 0.445). _VO2 kinetics parameters are presented in Table 2. Ap tended to increase with the swimming intensity (v) during the initial exercise, but differences were only noticed comparing 100 and 102.5%MLSS to 97.5%MLSS (F2,18 = 8.249, p< 0.05, p 2 = 0.478). Meanwhile, Ap was similar at final exercise for the three swim conditions (F2,18 = 1.167, p = 0.334, p 2 = 0.115). On the other hand, Ap decreased as a function of previous exercise for the three swims bouts. TDp, τp and MRT were similar as function of v at initial exercise and final exercise during the three swim- ming conditions. However, when analyzed the swim bouts as a function of previous exercise, TDp decreased for the 97.5%MLSS, but the values remained similar for 100 and 102.5%MLSS; τp decreased for all swim intensities, and MRT decreased for the 97.5 and 102.5%MLSS, but remained similar for 100%MLSS. The both measured _VO2baseline at initial exercise (F2,18 = 2.389, p = 0.120, p 2 = 0.210) and final exercise (F2,18 = 1.034, p = 0.376, p 2 = 0.103) were similar in between the three swim con- ditions, but _VO2baseline increased as a function of previous exercise (initial to final exercise) for all continuous intensities (F1,9 = 68.311, p< 0.001, p 2 = 0.884). Ac was similar as a function of v for both initial exercise (F2,18 = 0.134, p = 0.876, p 2 = 0.015) and final exercise (F2,18 = 1.974, p = 0.168, p 2 = 0.180). Moreover, at 97.5%MLSS, Ac was lower comparing initial and final exer- cise, but values remained similar for 100 and 102.5%MLSS. As of _VO2 kinetics was observed for all tested swimming intensities and testing phases (ini- tial and final exercise) only in two out of ten subjects. In one subject As was not observed. The As was observed for 6 swimmers during initial exercise and 8 swimmers during final exercise at 97.5%MLSS, for 6 swimmers during initial exercise and 7 swimmers during final exercise at 100%MLSS, and for 9 swimmers during initial exercise and 5 swimmers during final exercise Table 1. Mean (SD) values of swimming velocity (v), blood lactate concentrations ([La-]), heart rate (HR), and percentage of maximal oxygen uptake (%VO2max) are shown at 97.5, 100 and 102.5% of the maximal lactate steady state (MLSS) (N = 10). 97.5%MLSS 100%MLSS 102.5%MLSS v (m.s-1) 1.21 (0.07) 1.24 (0.07)1 1.27 (0.07)1,2 [La-] (mmol.L-1) 1.48 (0.39) 1.89 (0.77) 2.97 (0.87)1,2 HR (beats.min-1) 167.1 (15.0) 173.6 (9.7) 179.3 (9.2)1 % _VO2max (%) 78.9 (8.7) 84.7 (3.8)1 90.9 (4.6)1,2 1,2 Values different from 97.5 and 100%MLSS, respectively (p < 0.05). doi:10.1371/journal.pone.0167263.t001 _ Oxygen kinetics and aerobic endurance performance PLOS ONE | DOI:10.1371/journal.pone.0167263 February 28, 2017 5 / 12 at 102.5%MLSS. The As values are presented in Table 3. As tended to increase with swimming intensity during initial exercise, but keeping constant during final exercise whatever the inten- sity considered; however no statistical analysis was applied, once the occurrence of the As was apparently chaotic among swimmers both considering swimming intensities and phases of testing (initial and final exercise). The relative energy contribution for each one of the three swim intensity bouts is shown in Fig 1. The aerobic energy contribution decreased (F2,18 = 15.254, p< 0.001, p 2 = 0.629) and the anaerobic energy increased (F2,18 = 15.254, p< 0.001, p 2 = 0.629) at 102.5%MLSS compared to 97.5 and 100%MLSS. Discussion The purposes of this study were to examine the _VO2 kinetics responses during constant-veloc- ity swims at intensities of 97.5, 100 and 102.5%MLSS, the effect of previous exercise on the parameters of _VO2 kinetics, and the contribution of the energetic systems at the three Table 2. Mean (SD) values of VO2 kinetics parameters at velocities of 97.5, 100 and 102.5% of the maximal lactate steady state (MLSS) for the beginning of exercise until the break of swim for blood collection (initial exercise), and the second phase of exercise, starting after blood collec- tion (final exercise) (N = 10). 97.5%MLSS 100%MLSS 102.5%MLSS Initial exercise Final exercise Initial exercise Final exercise Initial exercise Final exercise VO2 baseline (mL.kg-1.min-1) 7.2 (2.1) 16.0 (5.3)a 6.0 (1.0) 17.4 (5.7)a 6.4 (0.8) 18.8 (5.8)a Ac (mL.kg-1.min-1) 16.4 (5.9) 10.4 (4.9)a 16.1 (7.1) 14.2 (5.4) 15.1 (6.5) 14.9 (5.7) Ap (mL.kg-1.min-1) 35.7 (7.3) 26.3 (7.4)a 41.0 (7.0)1 28.3 (5.2)a 41.3 (5.4)1 29.8 (5.5)a TDp (s) 14.3 (5.5) 12.0 (5.3)a 12.4 (8.1) 11.9 (4.9) 11.5 (6.8) 11.1 (4.7) τp (s) 16.3 (5.4) 10.8 (4.7)a 13.8 (4.5) 9.7 (4.5)a 16.0 (5.8) 9.6 (5.3)a MRT (s) 30.6 (5.2) 22.8 (5.4)a 26.2 (6.8) 21.6 (4.6) 27.4 (8.5) 20.7 (5.2)a Statistical analyses were described by intensity and previous exercise effect. 1 Values different from 97.5%MLSS for initial exercise. a Values different from initial exercise (p < 0.05). doi:10.1371/journal.pone.0167263.t002 Table 3. Individual and mean (SD) values of the amplitude of slow component (As) at velocities of 97.5, 100 and 102.5% of the maximal lactate steady state (MLSS) for the beginning of exercise until the break of swim for blood collection (initial exercise), and the second phase of exercise, starting after blood collection (final exercise) (N = 10). As (mL.kg-1.min-1) 97.5%MLSS 100%MLSS 102.5%MLSS swimmer Initial exercise Final exercise Initial exercise Final exercise Initial exercise Final exercise 1 1.7 2.9 2.3 3.8 4.5 1.6 2 2.3 0.7 4.4 0 3.7 0 3 1.1 0 2.6 0 4.4 0 4 0 0 0 0 0 0 5 4.2 0.9 2.8 0.8 2.9 0 6 0 0.8 0 0.8 1.9 2.2 7 0 1.2 0 0.9 7.2 1.1 8 0 1.7 2.8 1.1 4.5 0 9 2.5 1.3 2.6 1.5 6.1 0.8 10 1.4 0 0 1.5 5.1 1.8 Mean (SD) 2.2 (1.1) 1.4 (0.8) 2.9 (0.8) 1.5 (1.1) 4.5 (1.6) 1.5 (0.6) doi:10.1371/journal.pone.0167263.t003 _ Oxygen kinetics and aerobic endurance performance PLOS ONE | DOI:10.1371/journal.pone.0167263 February 28, 2017 6 / 12 conditions. The main original findings were that increasing exercise intensity resulted in greater primary amplitude of the _VO2 kinetics, in accordance with previous results in running [22]. As demonstrated by other studies [23–25], the previous exercise may increase the ampli- tude of the primary component and accelerate _VO2 kinetics (i.e., MRT) during the subsequent exercise. There was a significant increase in the anaerobic contribution when swimming above MLSS. However, the aerobic energetic system contribution corresponded to ~99% of the total energy demand of the exercise in all exercise conditions analyzed in this study. In sports science, _VO2 kinetics have added the understanding of physiological adjustments over time [2–4], such as muscle metabolism and systemic oxygen transport [26]. Moreover, one of the most relevant exercise intensities in swimming for aerobic training, prescription and evaluation is the v at which MLSS is obtained, being considered the direct and gold-stan- dard method for the evaluation of aerobic capacity [8, 10–12, 14]. Thus, both aspects ( _VO2 kinetics and MLSS) are decisive for the understanding of energy supply and oxidative metabo- lism supporting muscular exercise. Therefore, our purpose was to examine the amplitude and time adjustments of _VO2 kinetics during swims at intensities of 97.5, 100 and 102.5%MLSS, exploring the effects of small prescriptions variations on swimming oxidative physiology. The main findings were: (a) Ap tended to increase with swimming v for the initial phase of exercise, despite differences were only noticed comparing 100 and 102.5%MLSS to 97.5% MLSS. Meanwhile, Ap was similar at the final phase of exercise during the three swim condi- tions. However, Ap decreased as a function of previous exercise for the three swim intensities; (b) TDp, τp and MRT were similar irrespective of v both at initial and final exercise; (c) regard- ing the effect of previous exercise comparing initial and final exercise for the three swimming intensities, TDp decreased for the 97.5%MLSS, but was similar for 100 and 102.5%MLSS, τp decreased for all swim intensities, and MRT decreased for the 97.5 and 102.5%MLSS, but was similar for 100%MLSS; (d) although As was not evident for all swimmers during the three swimming conditions, it tended to increase with intensity during initial exercise, remaining constant during final exercise; (e) Ac was similar both for the initial and final exercise compar- ing the three swim intensities, but was lower during final exercise compared to initial exercise at 97.5%MLSS, and was similar at 100 and 102.5%MLSS; (f) aerobic and anaerobic energy con- tributions were different at 102.5%MLSS compared to lower swim velocities; (g) at the three Fig 1. Mean ± SD of aerobic and anaerobic energy relative contribution values at velocities corresponding to 97.5, 100 and 102.5% of the maximal lactate steady state (MLSS). doi:10.1371/journal.pone.0167263.g001 Oxygen kinetics and aerobic endurance performance PLOS ONE | DOI:10.1371/journal.pone.0167263 February 28, 2017 7 / 12 swim intensities, the aerobic contribution values were higher than 98% of the total energy input. The _VO2 values in the present study were directly measured breath-by-breath throughout time for the three swim intensities. Subsequently, the _VO2 data were fitted through mathemat- ical modelling as previously applied in swimming for maximal and submaximal exercises [15, 19, 27–30]. Some studies have reported _VO2 kinetics at intensities near the maximal v where a steady state in swimming is found (MLSS) [27–29], however we are unaware of a study that has evaluated and compared _VO2 kinetics at or around the MLSS in swimming. Most of previ- ous studies reported in sports science [2, 15, 19, 27–31] have studied _VO2 kinetics at maximal and submaximal intensities, demonstrating the fundamental role of _VO2 kinetics to under- stand the physiological mechanisms underpinning the dynamics of the aerobic response at dif- ferent exercise intensities. Thus, the understanding of the _VO2 kinetics throughout time may aid the evaluation of aerobic capacity and prescription of specific training sets during these fundamental training intensities around MLSS. The 100%MLSS v values reported in this study are in accordance with those reported in previous ones [13, 14, 32], in spite of the fact that most of the swimmers examined in the previ- ous studies were male when compared with the female subjects of the present study. Despite higher v values at a given relative intensity are expected to be higher for male than female counterparts of similar training level [33], the sex similitude comparing our results with litera- ture could likely be explained by a higher technical and biomechanical proficiency of our female swimmers when compared to the male swimmers of the previous studies. Indeed, the % _VO2max at 100%MLSS (85 ± 4%) observed in the present study for women is similar to previ- ously reported data for men (86.1% _VO2peak) [34], suggesting similar levels of aerobic capacity development, even the _VO2max= _VO2peak being higher in the previous study (mean = ~83 mL. kg-1.min-1) when compared with our results (54.9 ± 6.7 mL.kg-1.min-1). Meanwhile, the mean HR value at 100%MLSS was 174 ± 10 beats.min-1 in the present study, values which were simi- lar to the previous reported in literature [32, 34], as expected by the comparable age of samples. Moreover, the [La-] at 100%MLSS (1.89 ± 0.77 mmol.L-1) in the present study were lower when compared to swimming literature (2.8–3.3 mmol.L-1) [14, 34, 35]. These lower [La-] val- ues may be explained by sex differences for similar levels of aerobic capacity development, with expected lower values for women due to lower body mass and lean muscle mass com- pared to men [36]. Furthermore, women have showed lower testosterone concentration com- pared to men [37] during aerobic endurance exercise [33, 36], suggesting different metabolic contributions between carbohydrates and fat during long-distance exercise [33, 38], and sup- porting comparable lower [La-]. Since the early research on _VO2 kinetics [39] until up to date, the time constant (τ) has been studied in sports science in the attempt to comprehend the physiological adjustments during the non-steady state period at the beginning of exercise due to the increase of metabolic demand. In the present study, the τp values were similar between intensity levels for the initial exercise phase (mean = 15.4 ± 5.2 s) and final exercise phase (mean = 10.0 ± 4.7 s), but the val- ues decreased with previous exercise for the three swim conditions. This is particularly relevant for training practice, underlining the influence of previous exercise on the subsequent meta- bolic dynamics. In all studied exercise intensities, the τp in the present study showed similar values compared than those previously reported in swimming (~15–20 s) [27–29], cycling [40, 41], rowing [42], and running [43, 44]. Thus, those values reported for intensities up to and above the MLSS seem to behave similarly as expected, based on the previous knowledge on the Oxygen kinetics and aerobic endurance performance PLOS ONE | DOI:10.1371/journal.pone.0167263 February 28, 2017 8 / 12 _VO2 kinetics during different intensity domains for well-trained athletes. Indeed, a faster attainment of a steady state and a reduction in the oxygen deficit are associated to the fatigue delay and increase in exhaustion time, being well trained athletes able to perform at higher intensities with lower requirements of anaerobic energy during the transition from rest to exercise [5]. Hence, the lower τp values reported in this study when compared to previously published ones regarding physiological adaptations induced by aerobic endurance training confirm the highly endurance training status and specialization (endurance athletes) of our swimmers [5, 44]. Partially in contrast with previous literature that showed the existence of the As at these exercise intensities [2, 4, 5], in the present study it has shown to occur chaotically during the three swimming conditions, with very diverse individual occurrence profiles; however, observ- ing the sample data a tendency to As increase as a function of intensity was observed (2.2 ± 1.1, 2.9 ± 0.8 and 4.5 ± 1.6 mL.kg-1.min-1, respectively for 97.5, 100 and 102.5%MLSS), but only during initial exercise, not during the final phase after metabolic adaptation already occurred. Besides, only two swimmers showed As occurrence in all trials both at the initial and final exer- cise phases, and one swimmer did not show any As during all the swimming efforts and phases. It is worthy to emphasize the curiosity of that particular swimmer being a national record holder (800 and 1500m) and the best endurance swimmer of the sample. These partially con- tradictory findings could be explained, at least in part, by the specific physiological adaptations occurred through the highly endurance training status for our swimmers, such as faster τp [44], possible increase in the mitochondrial content of the cell [45], beyond also possible alter- ations in the mitochondrial sensitivity to the respiration regulators [46], and the fact of these endurance athletes might have mainly type I muscle fibers [45]. Thus, our endurance swim- mers with fast _VO2 kinetics would be able to adjust faster the physiological requirements for aerobic performance during the high intensity aerobic exercises, minimizing the As demand. In addition, the appearance of the As is normally explained by a phenomena that may be atten- uated in our very specialized sample, namely the recruitment of type II fibers with fatigue [47], after which the magnitude of As has been correlated with the rise of [La-] [2, 45]. Thereby, the absence of significant As in the present study may be likely explained by the high-level of endurance training of the sample [48]. Moreover, to reinforce the predominance of aerobic energy system during the three swim conditions around MLSS, the present study determined the total energy contribution at each one of the studied exercise intensities. At all swimming intensities up to and above MLSS, the aerobic energy contribution was higher than 98% of the total energy contribution; however there were significant differences between the highest and the lower v regarding aerobic and anaerobic energy contributions. This study was the first study to show the energy contribution during intensities at and around MLSS directly measured breath-by-breath in swimming, which highlights that even at intensities above MLSS; the total energy contribution was mainly and almost exclusively controlled by the oxidative bioenergetics system. Conclusions The present study showed that well-trained endurance swimmers with a fast component of _VO2 kinetics, i.e. an abrupt and fast increase in _VO2 response, and low [La-] may be able to adjust faster the physiological requirements during intensities up to and slightly above MLSS to minimize the appearance of the slow component of _VO2 and reduce the oxygen deficit, both parameters are associated to the fatigue delay and the increase in exhaustion time, key factors to endurance performance. however, these fast adjustments were not able to control the progressive fatigue occurred slightly above MLSS, and most of swimmers reached Oxygen kinetics and aerobic endurance performance PLOS ONE | DOI:10.1371/journal.pone.0167263 February 28, 2017 9 / 12 exhaustion before 30min swam. Moreover, the data shows that the aerobic energy contribution at intensities up to and slightly above MLSS plays a fundamental role controlling almost exclu- sively the required energy supply. Supporting information S1 File. Values of physiological parameters at 97.5, 100 and 102.5% of the maximal lactate steady state (MLSS) (N = 10). (PDF) S2 File. Values of VO2 kinetics parameters at 97.5, 100 and 102.5% of the maximal lactate steady state (MLSS) (N = 10). (PDF) Acknowledgments This investigation was supported by grants of the Capes Foundation, Ministry of Education of Brazil (BEX: 0536/10-5), and project PTDC/DES/101224/2008 (FCOMP-01-0124-FEDER- 009577). Author Contributions Conceptualization: JGP JPVB CCG RJF. Data curation: JGP LM JPVB CCG RJF. Formal analysis: JGP LM JPVB. Funding acquisition: JPVB RJF. Investigation: JGP JPVB CCG RJF LM. Methodology: JGP JPVB LM CCG RJF. Project administration: JGP JPVB RJF. Resources: JGP RJF JPVB. Software: LM JGP JPVB. Supervision: JGP JPVB CCG RJF LM. Validation: JGP LM JPVB CCG RJF. Visualization: JGP JPVB CCG RJF LM. Writing – original draft: JGP JPVB RJF CCG LM. Writing – review & editing: JGP JPVB CCG RJF LM. References 1. Bosquet L, Leger L, Legros P. Methods to determine aerobic endurance. Sports Med. 2002; 32 (11):675–700. PMID: 12196030 2. Gaesser GA, Poole DC. The slow component of oxygen uptake kinetics in humans. Exerc Sport Sci Rev. 1996; 24(1):35–71. 3. Poole DC, Ward SA, Gardner GW, Whipp BJ. Metabolic and respiratory profile of the upper limit for pro- longed exercise in man. Ergonomics. 1988; 31(9):1265–79. Epub 1988/09/01. doi: 10.1080/ 00140138808966766 PMID: 3191904 _ Oxygen kinetics and aerobic endurance performance PLOS ONE | DOI:10.1371/journal.pone.0167263 February 28, 2017 10 / 12 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0167263.s001 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0167263.s002 http://www.ncbi.nlm.nih.gov/pubmed/12196030 http://dx.doi.org/10.1080/00140138808966766 http://dx.doi.org/10.1080/00140138808966766 http://www.ncbi.nlm.nih.gov/pubmed/3191904 4. Poole DC, Richardson RS. Determinants of oxygen uptake. Implications for exercise testing. Sports Med. 1997; 24(5):308–20. Epub 1997/11/22. PMID: 9368277 5. Burnley M, Jones A. Oxygen uptake kinetics as a determinant of sports performance. European Journal of Sport Science. 2007; 7(2):63–9. 6. Whipp BJ, Wasserman K. Oxygen uptake kinetics for various intensities of constant-load work. J Appl Physiol. 1972; 33(3):351–6. Epub 1972/09/01. PMID: 5056210 7. Whipp BJ, Ward SA. Physiological determinants of pulmonary gas exchange kinetics during exercise. Med Sci Sports Exerc. 1990; 22(1):62–71. Epub 1990/02/01. PMID: 2406547 8. Beneke R. Maximal lactate steady state concentration (MLSS): experimental and modelling approaches. Eur J Appl Physiol. 2003; 88(4–5):361–9. doi: 10.1007/s00421-002-0713-2 PMID: 12527964 9. Heck H, Mader A, Hess G, Mucke S, Muller R, Hollmann W. Justification of the 4-mmol/l lactate thresh- old. Int J Sports Med. 1985; 6(3):117–30. doi: 10.1055/s-2008-1025824 PMID: 4030186 10. Beneke R, von Duvillard SP. Determination of maximal lactate steady state response in selected sports events. Med Sci Sports Exerc. 1996; 28(2):241–6. PMID: 8775160 11. Faude O, Kindermann W, Meyer T. Lactate threshold concepts: how valid are they? Sports Med. 2009; 39(6):469–90. doi: 10.2165/00007256-200939060-00003 PMID: 19453206 12. Billat VL, Sirvent P, Py G, Koralsztein JP, Mercier J. The concept of maximal lactate steady state: a bridge between biochemistry, physiology and sport science. Sports Med. 2003; 33(6):407–26. PMID: 12744715 13. Baron B, Dekerle J, Depretz S, Lefevre T, Pelayo P. Self selected speed and maximal lactate steady state speed in swimming. J Sports Med Phys Fitness. 2005; 45(1):1–6. PMID: 16208283 14. Pelarigo JG, Denadai BS, Greco CC. Stroke phases responses around maximal lactate steady state in front crawl. J Sci Med Sport. 2011; 14(2):168 e1–e5. Epub 2010/10/12. 15. Sousa AC, Vilas-Boas JP, Fernandes RJ. VO2 Kinetics and Metabolic Contributions Whilst Swimming at 95, 100, and 105% of the Velocity at VO2max. Biomed Res Int. 2014; 1(1):1–9. Epub 2014/07/22. 16. Fernandes RJ, Billat VL, Cruz AC, Colaco PJ, Cardoso CS, Vilas-Boas JP. Does net energy cost of swimming affect time to exhaustion at the individual’s maximal oxygen consumption velocity? J Sports Med Phys Fitness. 2006; 46(3):373–80. PMID: 16998440 17. Machado L, Almeida M, Morais P, Fernandes R, Vilas-Boas JP, editors. Assessing the individual anaer- obic threshold: the mathematical model. Xth International Symposium of Biomechanics and Medicine in Swimming; 2006; Porto, Portugal2006. 18. Baldari C, Fernandes RJ, Meucci M, Ribeiro J, Vilas-Boas JP, Guidetti L. Is the new AquaTrainer® snor- kel valid for VO2 assessment in swimming? Int J Sports Med. 2013; 34(4):336–44. doi: 10.1055/s- 0032-1321804 PMID: 23041962 19. Fernandes RJ, de Jesus K, Baldari C, Sousa AC, Vilas-Boas JP, Guidetti L. Different VO2max time- averaging intervals in swimming. Int J Sports Med. 2012; 33(12):1010–5. doi: 10.1055/s-0032-1316362 PMID: 22791619 20. di Prampero PE. Energetics of muscular exercise. Rev Physiol Biochem Pharmacol. 1981; 89(1):143– 222. Epub 1981/01/01. 21. Stevens JP. Applied multivariate statistics for the social sciences. Fourth edition ed. Mahwah: Law- rence Erlbaum Associates; 2002. 22. Carter H, Pringle JS, Jones AM, Doust JH. Oxygen uptake kinetics during treadmill running across exer- cise intensity domains. Eur J Appl Physiol. 2002; 86(4):347–54. PMID: 11990749 23. Burnley M, Doust JH, Carter H, Jones AM. Effects of prior exercise and recovery duration on oxygen uptake kinetics during heavy exercise in humans. Exp Physiol. 2001; 86(3):417–25. PMID: 11429659 24. Sousa A, Ribeiro J, Sousa M, Vilas-Boas JP, Fernandes RJ. Influence of Prior Exercise on VO2 Kinetics Subsequent Exhaustive Rowing Performance. PLoS One. 2014; 9(1):0084208. 25. Jones AM, Koppo K, Burnley M. Effects of prior exercise on metabolic and gas exchange responses to exercise. Sports Med. 2003; 33(13):949–71. PMID: 14606924 26. Xu F, Rhodes EC. Oxygen uptake kinetics during exercise. Sports Med. 1999; 27(5):313–27. PMID: 10368878 27. Reis JF, Alves FB, Bruno PM, Vleck V, Millet GP. Effects of aerobic fitness on oxygen uptake kinetics in heavy intensity swimming. Eur J Appl Physiol. 2012; 112(5):1689–97. doi: 10.1007/s00421-011-2126-6 PMID: 21879352 28. Reis JF, Alves FB, Bruno PM, Vleck V, Millet GP. Oxygen uptake kinetics and middle distance swim- ming performance. J Sci Med Sport. 2012; 15(1):58–63. Epub 2011/08/02. doi: 10.1016/j.jsams.2011. 05.012 PMID: 21802360 Oxygen kinetics and aerobic endurance performance PLOS ONE | DOI:10.1371/journal.pone.0167263 February 28, 2017 11 / 12 http://www.ncbi.nlm.nih.gov/pubmed/9368277 http://www.ncbi.nlm.nih.gov/pubmed/5056210 http://www.ncbi.nlm.nih.gov/pubmed/2406547 http://dx.doi.org/10.1007/s00421-002-0713-2 http://www.ncbi.nlm.nih.gov/pubmed/12527964 http://dx.doi.org/10.1055/s-2008-1025824 http://www.ncbi.nlm.nih.gov/pubmed/4030186 http://www.ncbi.nlm.nih.gov/pubmed/8775160 http://dx.doi.org/10.2165/00007256-200939060-00003 http://www.ncbi.nlm.nih.gov/pubmed/19453206 http://www.ncbi.nlm.nih.gov/pubmed/12744715 http://www.ncbi.nlm.nih.gov/pubmed/16208283 http://www.ncbi.nlm.nih.gov/pubmed/16998440 http://dx.doi.org/10.1055/s-0032-1321804 http://dx.doi.org/10.1055/s-0032-1321804 http://www.ncbi.nlm.nih.gov/pubmed/23041962 http://dx.doi.org/10.1055/s-0032-1316362 http://www.ncbi.nlm.nih.gov/pubmed/22791619 http://www.ncbi.nlm.nih.gov/pubmed/11990749 http://www.ncbi.nlm.nih.gov/pubmed/11429659 http://www.ncbi.nlm.nih.gov/pubmed/14606924 http://www.ncbi.nlm.nih.gov/pubmed/10368878 http://dx.doi.org/10.1007/s00421-011-2126-6 http://www.ncbi.nlm.nih.gov/pubmed/21879352 http://dx.doi.org/10.1016/j.jsams.2011.05.012 http://dx.doi.org/10.1016/j.jsams.2011.05.012 http://www.ncbi.nlm.nih.gov/pubmed/21802360 29. Pessoa Filho DM, Alves FB, Reis JF, Greco CC, Denadai BS. VO2 kinetics during heavy and severe exercise in swimming. Int J Sports Med. 2012; 33(9):744–8. doi: 10.1055/s-0031-1299753 PMID: 22592546 30. Reis VM, Marinho DA, Policarpo FB, Carneiro AL, Baldari C, Silva AJ. Examining the accumulated oxy- gen deficit method in front crawl swimming. Int J Sports Med. 2010; 31(6):421–7. doi: 10.1055/s-0030- 1248286 PMID: 20301045 31. Jones AM, Burnley M. Oxygen uptake kinetics: an underappreciated determinant of exercise perfor- mance. Int J Sports Physiol Perform. 2009; 4(4):524–32. PMID: 20029103 32. Dekerle J, Pelayo P, Clipet B, Depretz S, Lefevre T, Sidney M. Critical swimming speed does not repre- sent the speed at maximal lactate steady state. Int J Sports Med. 2005; 26(7):524–30. doi: 10.1055/s- 2004-821227 PMID: 16195984 33. Greco CC, Pelarigo JG, Figueira TR, Denadai BS. Effects of gender on stroke rates, critical speed and velocity of a 30-min swim in young swimmers. J Sports Sci Med. 2007; 6(4):441–7. PMID: 24149476 34. Dekerle J, Nesi X, Lefevre T, Depretz S, Sidney M, Marchand FH, et al. Stroking parameters in front crawl swimming and maximal lactate steady state speed. Int J Sports Med. 2005; 26(1):53–8. doi: 10. 1055/s-2004-817854 PMID: 15643535 35. Figueiredo P, Nazário R, Sousa M, Pelarigo JG, Vilas-Boas JP, Fernandes R. Kinematical Analysis along Maximal Lactate Steady State Swimming Intensity. J Sports Sci Med. 2014; 13(3):610–5. PMID: 25177189 36. Crewther B, Cronin J, Keogh J. Possible stimuli for strength and power adaptation: acute metabolic responses. Sports Med. 2006; 36(1):65–78. PMID: 16445311 37. Deschenes MR, Kraemer WJ. Performance and physiologic adaptations to resistance training. Am J Phys Med Rehabil. 2002; 81(11 Suppl):S3–16. Epub 2002/11/01. doi: 10.1097/01.PHM.0000029722. 06777.E9 PMID: 12409807 38. Tarnopolsky MA, Atkinson SA, Phillips SM, MacDougall JD. Carbohydrate loading and metabolism dur- ing exercise in men and women. J Appl Physiol. 1995; 78(4):1360–8. Epub 1995/04/01. PMID: 7615443 39. Margaria R, Edwards HT, Dill DB. The possible mechanisms of contracting and paying the oxygen debt and the rôle of lactic acid in muscular contraction1933 1933-11-30 00:00:00. 689–715 p. 40. Berger NJ, Jones AM. Pulmonary O2 uptake on-kinetics in sprint- and endurance-trained athletes. Appl Physiol Nutr Metab. 2007; 32(3):383–93. Epub 2007/05/19. doi: 10.1139/H06-109 PMID: 17510672 41. Koppo K, Bouckaert J, Jones AM. Effects of training status and exercise intensity on phase II VO2 kinet- ics. Med Sci Sports Exerc. 2004; 36(2):225–32. Epub 2004/02/10. doi: 10.1249/01.MSS.0000113473. 48220.20 PMID: 14767244 42. Ingham SA, Carter H, Whyte GP, Doust JH. Comparison of the oxygen uptake kinetics of club and olym- pic champion rowers. Med Sci Sports Exerc. 2007; 39(5):865–71. Epub 2007/05/01. doi: 10.1249/mss. 0b013e31803350c7 PMID: 17468587 43. Borrani F, Candau R, Millet GY, Perrey S, Fuchslocher J, Rouillon JD. Is the VO2 slow component dependent on progressive recruitment of fast-twitch fibers in trained runners? J Appl Physiol. 2001; 90 (6):2212–20. PMID: 11356785 44. Carter H, Jones AM, Barstow TJ, Burnley M, Williams C, Doust JH. Effect of endurance training on oxy- gen uptake kinetics during treadmill running. J Appl Physiol. 2000; 89(5):1744–52. PMID: 11053321 45. Holloszy JO, Coyle EF. Adaptations of skeletal muscle to endurance exercise and their metabolic con- sequences. J Appl Physiol Respir Environ Exerc Physiol. 1984; 56(4):831–8. Epub 1984/04/01. PMID: 6373687 46. Dudley GA, Tullson PC, Terjung RL. Influence of mitochondrial content on the sensitivity of respiratory control. J Biol Chem. 1987; 262(19):9109–14. Epub 1987/07/05. PMID: 3597408 47. Poole DC, Schaffartzik W, Knight DR, Derion T, Kennedy B, Guy HJ, et al. Contribution of exercising legs to the slow component of oxygen uptake kinetics in humans. J Appl Physiol. 1991; 71(4):1245–60. Epub 1991/10/01. PMID: 1757346 48. Billat V, Binsse V, Petit B, Koralsztein JP. High level runners are able to maintain a VO2 steady-state below VO2max in an all-out run over their critical velocity. Arch Physiol Biochem. 1998; 106(1):38–45. Epub 1998/10/23. doi: 10.1076/apab.106.1.38.4396 PMID: 9783059 Oxygen kinetics and aerobic endurance performance PLOS ONE | DOI:10.1371/journal.pone.0167263 February 28, 2017 12 / 12 http://dx.doi.org/10.1055/s-0031-1299753 http://www.ncbi.nlm.nih.gov/pubmed/22592546 http://dx.doi.org/10.1055/s-0030-1248286 http://dx.doi.org/10.1055/s-0030-1248286 http://www.ncbi.nlm.nih.gov/pubmed/20301045 http://www.ncbi.nlm.nih.gov/pubmed/20029103 http://dx.doi.org/10.1055/s-2004-821227 http://dx.doi.org/10.1055/s-2004-821227 http://www.ncbi.nlm.nih.gov/pubmed/16195984 http://www.ncbi.nlm.nih.gov/pubmed/24149476 http://dx.doi.org/10.1055/s-2004-817854 http://dx.doi.org/10.1055/s-2004-817854 http://www.ncbi.nlm.nih.gov/pubmed/15643535 http://www.ncbi.nlm.nih.gov/pubmed/25177189 http://www.ncbi.nlm.nih.gov/pubmed/16445311 http://dx.doi.org/10.1097/01.PHM.0000029722.06777.E9 http://dx.doi.org/10.1097/01.PHM.0000029722.06777.E9 http://www.ncbi.nlm.nih.gov/pubmed/12409807 http://www.ncbi.nlm.nih.gov/pubmed/7615443 http://dx.doi.org/10.1139/H06-109 http://www.ncbi.nlm.nih.gov/pubmed/17510672 http://dx.doi.org/10.1249/01.MSS.0000113473.48220.20 http://dx.doi.org/10.1249/01.MSS.0000113473.48220.20 http://www.ncbi.nlm.nih.gov/pubmed/14767244 http://dx.doi.org/10.1249/mss.0b013e31803350c7 http://dx.doi.org/10.1249/mss.0b013e31803350c7 http://www.ncbi.nlm.nih.gov/pubmed/17468587 http://www.ncbi.nlm.nih.gov/pubmed/11356785 http://www.ncbi.nlm.nih.gov/pubmed/11053321 http://www.ncbi.nlm.nih.gov/pubmed/6373687 http://www.ncbi.nlm.nih.gov/pubmed/3597408 http://www.ncbi.nlm.nih.gov/pubmed/1757346 http://dx.doi.org/10.1076/apab.106.1.38.4396 http://www.ncbi.nlm.nih.gov/pubmed/9783059