A “Blood Relationship” Between the Overlooked Minimum Lactate Equivalent and Maximal Lactate Steady State in Trained Runners. Back to the Old Days?

ORIGINAL RESEARCH published: 31 July 2018 doi: 10.3389/fphys.2018.01034 Frontiers in Physiology | www.frontiersin.org 1July 2018 | Volume 9 | Article 1034 Edited by: Hassane Zouhal, University of Rennes 2 – Upper Brittany, France Reviewed by: Daniel A. Kane, St. Francis Xavier University, Canada Thierry Busso, University of Saint-Etienne, France *Correspondence: Ibai Garcia-Tabar [email protected] Specialty section: This article was submitted to Exercise Physiology, a section of the journal Frontiers in Physiology Received: 22 May 2018 Accepted: 11 July 2018 Published: 31 July 2018 Citation: Garcia-Tabar I and Gorostiaga EM (2018) A “Blood Relationship” Between the Overlooked Minimum Lactate Equivalent and Maximal Lactate Steady State in Trained Runners. Back to the Old Days? Front. Physiol. 9:1034. doi: 10.3389/fphys.2018.01034 A“Blood Relationship” Between the Overlooked Minimum Lactate Equivalent and Maximal Lactate Steady State in Trained Runners. Back to the Old Days? Ibai Garcia-Tabar*and Esteban M. Gorostiaga Studies, Research and Sports Medicine Center, Government of Navarre, Pamplona, Spain Maximal Lactate Steady State (MLSS) and Lactate Threshold (LT) are physiologically-related and fundamental concepts within the sports and exercise sciences. Literature supporting their relationship, however, is scarce. Among the recognized LTs, we were particularly interested in the disused “Minimum Lactate Equivalent” (LEmin), first described in the early 1980s. We hypothesized that velocity at LT, conceptually comprehended as in the old days (LEmin), could predict velocity at MLSS (VMLSS) more accurate than some other blood lactate-related thresholds (BLRTs) routinely used nowadays by many sport science practitioners. Thirteen male endurance-trained [VMLSS 15.0 ±1.1 km·h−1; maximal oxygen uptake ( ˙ VO2max) 67.6 ±4.1 ml·kg−1·min−1] homogeneous (coefficient of variation: ≈7%) runners conducted 1) a submaximal discontinuous incremental running test to determine several BLRTs followed by a maximal ramp incremental running test for ˙ VO2max determination, and 2) several (4–5) constant velocity running tests to determine VMLSS with a precision of 0.20 km·h−1. Determined BLRTs include LEmin and LEmin-related LEmin plus 1 (LEmin+1 mM) and 1.5 mmol·L−1(LEmin+1.5 mM), along with well-established BLRTs such as conventionally-calculated LT, Dmax and fixed blood lactate concentration thresholds. LEmin did not differ from LT (P=0.71; ES: 0.08) and was 27% lower than MLSS (P<0.001; ES: 3.54). LEmin+1 mM was not different from MLSS (P=0.47; ES: 0.09). LEmin was the best predictor of VMLSS (r=0.91; P<0.001; SEE =0.47 km·h−1), followed by LEmin+1 mM (r=0.86; P<0.001; SEE =0.58 km·h−1) and LEmin+1.5 mM (r=0.84; P<0.001; SEE =0.86 km·h−1). There was no statistical difference between MLSS and estimated MLSS using LEmin prediction formula (P=0.99; ES: 0.001). Mean bias and limits of agreement were 0.00 ±0.45 km·h−1and ±0.89 km·h−1. Additionally, LEmin, LEmin+1 mM and LEmin+1.5 mM were the best predictors of ˙ VO2max (r=0.72–0.79; P<0.001). These results support LEmin, an objective submaximal overlooked and underused BLRT, to be one of the best single MLSS predictors in endurance trained runners. Our study advocates factors controlling LEmin to be shared, at least partly, with those controlling MLSS. Keywords: lactate threshold, aerobic capacity, Owles’ point, oxygen endurance performance limit, aerobic threshold, anaerobic threshold, endurance assessment, submaximal exercise testing Garcia-Tabar and Gorostiaga Lactate Equivalent (LE) vs. MLSS INTRODUCTION The exercise intensity corresponding to the maximal lactate steady state (MLSS) is a consistent physiological phenomenon describing the highest constant velocity or power output that can be maintained over time without continual blood lactate concentration (BLC) accumulation (Beneke, 1995). Nowadays MLSS is considered the gold standard endurance performance marker among the vast majority of sport and exercise science physiologists (Beneke, 1995; Llodio et al., 2016; Messias et al., 2017). MLSS is valuable, and more sensitive than maximal oxygen uptake ( ˙ VO2max), to diagnose endurance performance (Coyle et al., 1988), guide aerobic training (Haverty et al., 1988), evaluate endurance training-induced adaptations (Philp et al., 2008) and predict endurance performance (Haverty et al., 1988; Jones and Doust, 1998). Determination of MLSS is, however, cumbersome and interferes with the athlete’s training program since it requires several (3–6) constant workload tests on separate days lengthening aerobic conditioning evaluation to a minimum of 1–3 week period (Heck et al., 1985). In an attempt to overcome the shortcomings of multiple-day testing, simpler methods have been proposed to estimate MLSS from a single-day test, involving the use of either BLC-based measurements or some other bloodless simple measurements such as the peak workload reached during an incremental maximal test. Numerous studies conducted on competitive athletes have shown that the intensities corresponding to some blood lactate-related thresholds (BLRTs), such as the Onset of Blood Lactate Accumulation (OBLA) (Beneke, 1995; Van Schuylenbergh et al., 2004), Individual Anaerobic Threshold (IAT) (Beneke, 1995), Dmax (Van Schuylenbergh et al., 2004) or the Lactate Minimum Test (LMT) (Jones and Doust, 1998), predict MLSS with a wide range of correlation magnitudes (r=0.61–0.85). However, these correlation magnitudes are equal, or even lower, than the ones reported in those same studies when the peak workload attained during an incremental maximal test was used as MLSS predictor (r=0.85–0.94). Before the appearance of the MLSS concept and based on the early works of Barr and Himwich (1923) and Owles (1930) published in the 1920s, several researchers independently found that during graded incremental exercise there is a critical exercise intensity level unique to each individual above which BLC initiates to increase beyond resting values. In the following years this critical workload level, which always occurs at lower intensities than MLSS (Lehmann et al., 1983; Aunola and Rusko, 1988; Faude et al., 2009; Ferguson et al., 2018) and is frequently called “Lactate Threshold (LT)” (Jones and Ehrsam, 1982) [although it has also been termed “Owles’ Point” (Jones and Ehrsam, 1982), “Oxygen Endurance Performance Limit” (Hollmann, 1985), “Aerobic Threshold” (Kindermann et al., 1978) or “Anaerobic Threshold” (Wasserman et al., 1973)], was widely considered as the standard criterion measure to determine aerobic capacity (Weltman et al., 1987; Mezzani et al., 2012), predict endurance performance (Yoshida et al., 1990), and design endurance exercise training programs (Weltman et al., 1990); turning LT into a pivotal concept within the sports medicine and exercise sciences. Notwithstanding, there are still some relevant methodological limitations on the accurate and rigorous determination of LT, mainly when (a) it is determined by simple visual inspection of BLC-data plotted against workload due to the subjectivity of the analysis and poor inter-viewer and intermethod agreement (Yeh et al., 1983), (b) the initial workload and subsequent initial workload increments are not low enough to allow a preliminary BLC-baseline phase on the BLC kinetics during the graded exercise (Hollmann, 1985), and (c) the BLCdata-point interval is too large to detect LT with a suitable sensitivity (Hollmann, 1985). Beyond a shadow of a doubt, objective methodological approaches and appropriate rigorous protocols are needed to overcome these limitations (Brooks, 1985). Despite MLSS and LT being physiologically different, but probably related, fundamental concepts within the sports and exercise sciences (Ferguson et al., 2018), literature concerning their relationship is scarce. As far as the authors are aware, whether the velocity at LT (VLT) obtained during an incremental exercise test predicts the velocity at MLSS (VMLSS) in endurance trained runners has not been fully explored, and deserves further attention. We hypothesized that VLT, conceptually comprehended as in the old days (Owles, 1930), could predict VMLSS more accurate than some others BLRTs used nowadays by many authors and other sport science practitioners. Accordingly, the primary purpose of this study was to determine the applicability of the classical gold standard vLT,calculated objectively and in a standardized manner, to predict VMLSS in comparison with some other more commonly used parameters of BLC changes during incremental exercise in a homogeneous group of endurance trained runners. Among the recognized BLRTs (Faude et al., 2009) we were particularly interested in the “Minimum Lactate Equivalent” (LEmin), initially described by German authors in the early 1980s (Berg et al., 1980; Lehmann et al., 1983). LEmin is the minimum value of the BLC/workload vs. workload curve fitting during an incremental exercises test. Using an appropriate protocol with adequate opening and incremental workloads, the incremental test produces an idiosyncratic “U-shaped” curve fitting profile allowing mathematical impartial location of the transition at VLT with a very fine resolution. This seldom used method (LEmin) should not be confused with the much more popular “Lactate Minimum Test” (LMT), which was originally described by Tegtbur et al. (1993) and uses a preliminary relatively high level of exertion phase (hyperlactatemia phase) to setup the mentioned “U-shaped” curve fitting profile hampering heart rate (HR) data interpretation, and therefore, its on-field application. A secondary purpose of this study was to determine the extent to which some variables not requiring blood sampling, such as ˙ VO2max, peak treadmill velocity (PTV) or the velocity corresponding to the 90% of maximal heart rate (V90) (GarciaTabar et al., 2015b), are of potential interest to estimate VMLSS. To the best of our knowledge literature concerning VMLSS prediction from such variables in well-trained endurance runners is limited. Assessment and monitoring of aerobic capacity in this kind of athletes is of paramount importance (Halson, 2014), and consequently, this study has the potential to contribute with Frontiers in Physiology | www.frontiersin.org 2July 2018 | Volume 9 | Article 1034 Garcia-Tabar and Gorostiaga Lactate Equivalent (LE) vs. MLSS noteworthy scientific-based practical endurance performance implications. MATERIALS AND METHODS Subjects Fifteen male trained middleand long-distance runners were recruited from regional athletic clubs. Runners were required to meet the following inclusion criteria: (1) being male runners aged between 18 and 40; (2) having a VMLSS >13 km·h−1, and (3) a training routine of ≥3 aerobic running training sessions per week. Exclusions criteria were: (1) being taking any medication/supplementation that could affect BLC or HR values and (2) having any known cardiovascular, respiratory or circulatory dysfunction. One runner withdrew from the study due to personal reasons and another runner did not meet the inclusion criteria. Thirteen runners completed the study. Mean (±SD) age, height, body mass and percentage of body fat of the thirteen participants were 28 ±7 y, 1.76 ±0.05 m, 68.8 ±6.8 kg and 8.8 ±3.1%, respectively. Runners competed in races ranging from 800-m to half-marathon. The study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures were approved by the local Institutional Review Committee of the Instituto Navarro del Deporte y Jueventud (Government of Navarre, Spain). Inclusion and exclusion criteria, experimental rationale, testing procedures and associated risks and benefits of participation were fully explained to participants and their coaches by an oral presentation. Prior to any testing, participants acknowledged voluntary participation through written informed consent. Study Design A predictive cross-sectional study was conducted to determine VMLSS from a single-session submaximal discontinuous incremental running test (SD-IRT). Participants conducted 7–8 laboratory testing sessions. (1) Heath screening session: a maximal ramp incremental cycling test to discard any cardiovascular anomaly (12-lead electrocardiogram, GE Healthcare, CASE Marquette, Germany). (2) Familiarization session: a SD-IRT to accustom to the testing treadmill running protocol. This session was also utilized for anthropometric evaluation. (3) BLRTs and ˙ VO2max testing session: the SD-IRT previously used in the familiarization session to determine BLRTs, followed by a maximal ramp incremental running test (MR-IRT) to determine ˙ VO2max. (4) VMLSS testing: 4–5 constant velocity running tests (CVRTs) for VMLSS determination. Testing Procedures Participants were required to complete the study within 6 weeks. Testing sessions were performed at the same time of the day to lessen circadian variability, were preceded by 2 days of rest or very light exercise [<90 min at <70% maximal HR (HRmax)] and were separated from the last competitive race by ≥4 days to allow restoration of muscle glycogen. Runners arrived to each testing session in a rested and fully hydrated state, 2 h postprandial, having abstained from caffeinated and alcoholic beverages during the day. Subjects were asked to replicate diet and exercise regimens the 2 days preceding each testing session to limit fluctuations of initial glycogen concentration between trials (Van Schuylenbergh et al., 2004; Philp et al., 2008). Participants recorded their exercise training and diet throughout the experimental phase of the study on training and diet logs designed and provided by the authors. These detailed exercise and diet logs served to confirm fulfillment of diet and exercise instructions given for the 2 days preceding each testing session, and verified that only minor changes in training and aerobic conditioning occurred during the study (Farrell et al., 1979). Participants wore the same running trainers on each experimental day. Testing took place during May-June, i.e., beginning of the outdoor competitive season. All procedures were conducted on the same running ergometer (Kuntaväline, Hyper Treadmill 2040, Finland) with the gradient set at 1%, under temperature (22.3 ±1.4◦C), humidity (33 ±4%) and luminosity controlled laboratory conditions. BLRTs and ˙ VO2max Testing Athletes performed a SD-IRT for BLRTs determination, followed by a MR-IRT to determine their ˙ VO2max. The submaximal trial began at 7 km·h−1. Speed was increased by 1 km·h−1 every 2-min, with 1-min intervals between stages until a BLC ≥3 mmol·L−1was observed. On the basis that 1to 4-min stage duration protocols do not notably affect BLRTs detection (Yoshida, 1984), 2-min duration stages were chosen not to unnecessary lengthen the SD-IRT according to previous LEmin detection protocols (Berg et al., 1980, 1990; Lehmann et al., 1983; Aunola and Rusko, 1988). Immediately after each stage, capillary blood samples for BLC measurements were obtained. After a 10-min rest, subjects began the MR-IRT. Initial speed was 10 km·h−1and was increased by 1 km·h−1every min until volitional exhaustion. Volunteers were vigorously encouraged to complete exhaustion. Post-exercise capillary blood samples after 3 min of passive recovery were obtained for peak BLC (BLCpeak) determination. HR during both trials (Polar Electro Oy, RS800CX, Finland) and metabolic data during the MRIRT (Vista Mini-CPX, Vacu-Med, Silver Edition 17670, Ventura, CA, USA) were monitored and averaged over 30-s. PTV, HRmax (Garcia-Tabar et al., 2017) and ˙ VO2max (Garcia-Tabar et al., 2015a) were determined following procedures previously described. VMLSS Testing On subsequent laboratory visits, runners completed 4–5 CVRTs. Each CVRT consisted of 30-min running at the selected speeds with 1-min interruptions every 10-min for blood sampling (i.e., 32-min duration CVRTs). Capillary blood samples were obtained at rest, and at min 10, 21 and 32 of exercise. An increase in BLC <1.0 mmol·L−1during the last 20 min of exercise (i.e., between the 10th and the 32nd min of the CVRT) was defined as the criterion for BLC to be considered at a steady state (Beneke, 1995). VMLSS was defined as the highest running velocity meeting this stability criterion. Running velocity of the first CVRT corresponded to approximately 80% of the PTV achieved during the maximal trial. Depending on the BLC stability of Frontiers in Physiology | www.frontiersin.org 3July 2018 | Volume 9 | Article 1034 Garcia-Tabar and Gorostiaga Lactate Equivalent (LE) vs. MLSS this first CVRT, the velocity was increased or decreased in the following CVRTs. If during the first CVRT a steady state or decrease in BLC was found, the velocity for the next CVRT was increased by 0.4 km·h−1. Conversely, if an increase in BLC superior to the stability criterion was observed, running velocity for the next CVRT was decreased by 0.4 km·h−1. This process of increasing or decreasing running velocity by 0.4 km·h−1, and later by 0.2 km·h−1, was further repeated in subsequent tests until VMLSS was determined with a precision of 0.2 km·h−1. HR was monitored and averaged as abovementioned. Blood Sampling and Blood Lactate Concentration (BLC) Determination A hyperemic earlobe was cleaned and dried before puncturing by a lancet device to aspirate a 5 µL whole blood sample into an enzyme-coated electrode test strip. BLC was determined via amperometric measurement using a portable analyzer (Arkray KDK Corporation, Lactate Pro LT-1710, Shiga, Japan) calibrated before every test. Manufacturers report coefficients of variation (CVs) of 3.2 and 2.6% for lactate standards of 2 and 11 mmol·L−1, respectively. Determination of Blood Lactate-Related Thresholds (BLRTs) Nine different BLRTs were determined. LT0.2 mM and LT1. LT0.2 mM was defined as the stage prior to a ≥0.2 mmol·L−1 BLC elevation above baseline values (Stratton et al., 2009). To overcome the error associated with the analyzer (Weltman et al., 1987), the highest stage above which BLC increased by ≥0.1 mmol·L−1in the following stage and ≥0.2 mmol·L−1 in the subsequent stage was also chosen as a threshold and named LT1. LEmin, LEmin+1 mM and LEmin+1.5 mM. The velocity corresponding to the Minimum Lactate Equivalent (VLEmin) (Berg et al., 1990) was considered the minimum value of the quotient BLC/velocity in the individual BLC/velocity vs. velocity second-order polynomial curves. Velocity associated with the Minimum Lactate Equivalent plus 1 (VLEmin+1 mM) and 1.5 mmol·L−1(VLEmin+1.5 mM) were defined as the running velocities at 1.0 and 1.5 mmol·L−1above VLEmin in the individual BLC vs. velocity second-order polynomial curves, respectively. Dmax. Velocity at Dmax was considered the maximum perpendicular distance from the straight line between the first and final BLC data-points to the third-order polynomial curve describing the BLC kinetics during the SD-IRT (Cheng et al., 1992). Fixed blood lactate concentration (FBLC) thresholds. Velocities at FBLC thresholds of 2 (FBLC2 mM), 2.5 (FBLC2.5 mM) and 3 mmol·L−1(FBLC3 mM) commonly use in real practice (Seiler, 2010; Garcia-Tabar et al., 2017) were determined from the individual BLC vs. velocity second-order polynomial curves. Determination of BLRTs is illustrated in Figure 1. Velocities at the BLRTs were determined using MATLAB R2015a (The MathWorks Inc., Natick, MA, USA). Coefficients of determination (R2) of the individual secondand third-order BLC vs. velocity and second-order BLC/velocity quotient vs. velocity polynomial curves were all >0.90. Velocities at BLRTs (Weltman et al., 1990), as well as VMLSS (Hauser et al., 2013), frequently show test-retest intraclass correlation coefficients >0.94, and CVs ≤3%. HR values at the BLRTs were computed from the individual HR vs. velocity linear regression equations (r>0.98; P<0.001). V90 was also calculated from the individual linear HR vs. velocity regressions obtained during the SD-IRTs (Garcia-Tabar et al., 2017). Statistics Standard statistical methods were used for the calculation of means, standard deviations (SD), standard errors of the estimates (SEE) and confidence intervals (CI). Data were analyzed using parametric statistics following confirmation of normality (Kolmogorov–Smirnov test), homoscedasticity (Levene’s test), and when appropriate sphericity (Mauchly’s test). The Greenhouse-Geisser correction factor to reduce the risk of type I error was applied where sphericity assumptions were violated. Student’s paired t-tests were used to evaluate differences between each BLRT with MLSS. The magnitudes of the differences were assessed using 90% CI and Hedges’ geffect sizes (ES) (Hedges, 1981). Differences were considered nonsubstantial if the 90% CIs overlapped zero. ES values of 0.2, 0.5, and >0.8 were considered to represent small, moderate, and large differences, respectively. Differences in BLC and HR along the CVRTs were identified by one-way repeated measures ANOVA with Bonferroni correction for multiple comparisons. Two-factorial ANOVA with the Scheffé post-hoc test was used to identify differences in BLC and HR between the CVRTs at VMLSS and at 0.2 km·h−1above VMLSS (VMLSS+0.2). Linear regression analyses with Pearson’s correlation coefficients (r) were performed to determine the relationships between the variables of interest. When pertinent, slopes of regression lines were compared using analysis of covariance (ANCOVA). Agreement with the reference method (VMLSS) was assessed by mean bias and limits of agreement (LOAs) (Krouwer, 2008). Post-hoc power calculation for the linear regressions, assuming type I error of 0.05, indicated a power >99%. Analyses were performed using IMB SPSS Statistics 22 (IBM Corporation, NY, USA). Significance was set at P<0.05 for the analyses that did not require post-hoc adjustment. Descriptive statistics are reported as means (±SD). RESULTS BLRTs and ˙ VO2max Testing The SD-IRT lasted 32:00 ±4:24 min:s. Runners achieved a treadmill velocity of 17.0 ±1.5 km·h−1(range 15.0–19.0). BLC and %HRmax at completion of the SD-IRT were 3.4 ±0.6 mmol·L−1(range 3.0–5.4) and 92 ±2% (range 87– 93), respectively. Figure 2 depicts BLC and %HRmax pattern responses to the SD-IRT. Descriptive characteristics of the BLRTs are depicted in Table 1. BLC resting values prior to the beginning of the MR-IRT were 1.1 ±0.2 mmol·L−1(range 0.8–1.7). Table 2 elucidated the maximal nature of the MR-IRT. VMLSS Testing Descriptive features of the MLSS are displayed along with the BLRTs (Table 1). Velocity at LEmin did not differ from that at Frontiers in Physiology | www.frontiersin.org 4July 2018 | Volume 9 | Article 1034 Garcia-Tabar and Gorostiaga Lactate Equivalent (LE) vs. MLSS FIGURE 1 | Illustration of blood lactate-related thresholds (BLRTs) determination in a representative participant. Dashed lines: second-order polynomial curve fits. Dotted lines: the greatest perpendicular distance from the third-order polynomial BLC-velocity curve fit to the generated straight line by the two end data-points of this curve. Note that for clearness of figure presentation, Dmax determination is illustrated together with the rest of BLRTs on a second-order polynomial curve fit, although actually it was determined on third-order curvilinear fits as originally described (Cheng et al., 1992). LT1(P=0.71; 90% CI: −0.74 to 0.47; ES: 0.08) and was 27% lower than VMLSS (P<0.001; 90% CI: −3.80 to −2.83; ES: 3.54). Velocities at FBLC2 mM (P=0.50; 90% CI: −0.69 to 0.30; ES: 0.15) and LEmin+1 mM (P=0.47; 90% CI: −0.17 to 0.42; ES: 0.09) were not different from VMLSS. V90 (16.1 ±1.2 km·h−1, range 13.9–18.1) was 1.1 km·h−1(7%) higher than VMLSS (P<0.001; 90% CI: 0.77–1.53; ES: 0.96), and not different from velocity at FBLC2.5 mM (P=0.619; 90% CI: −0.27 to 0.49; ES: 0.09) and VLEmin+1.5 mM (P=0.543; 90% CI: −0.42 to 0.87; ES: 0.15). HR associated with VMLSS during the SD-IRT was 86 ±5% HRmax, and was not different from %HRmax at FBLC2 mM (P=0.93; 90% CI: −2.99 to 3.29; ES: 0.01), LEmin+1 mM (P=0.92; 90% CI: −2.56 to 2.86; ES: 0.04) and FBLC2.5 mM (P=0.08; 90% CI: 0.25–6.55; ES: 0.78). BLC and %HRmax responses to the CVRTs performed at VMLSS and at VMLSS+0.2 are illustrated in Figure 3. One runner exhausted at min 29 of the CVRT at Frontiers in Physiology | www.frontiersin.org 5July 2018 | Volume 9 | Article 1034 Garcia-Tabar and Gorostiaga Lactate Equivalent (LE) vs. MLSS FIGURE 2 | Mean (SD) blood lactate and heart rate responses to the submaximal discontinuous incremental running exercise test. All subjects terminated the 15 km·h−1exercise stage. Mean (SD) values at completion of the test of subjects achieving ≥16 km·h−1are indicated by dashed lines. TABLE 1 | Descriptive features of the determined blood lactate-related thresholds and maximal lactate steady state (MLSS) (n=13). km·h−1%MLSSV%PTV %HRmax Mean ±SD Range Mean ±SD Range Mean ±SD Range Mean ±SD Range LEmin 11.6 ±0.8** 10.5–12.6 77 ±2** 74–80 58 ±3** 55–64 75 ±5** 63–80 LT111.7 ±1.7** 9.0–14.0 78 ±7** 64–88 59 ±7** 47–70 76 ±4** 66–83 LT0.2 mM 12.5 ±1.4** 10.0–15.0 84 ±6** 73–94 63 ±5** 52–70 79 ±3** 70–83 Dmax 13.2 ±1.2** 11.5–14.6 87 ±4** 81–95 66 ±4** 59–73 81 ±2** 77–85 FBLC2 mM 14.8 ±1.5 12.8–17.0 99 ±7 84–107 75 ±5 67–82 86 ±3* 80–90 MLSS 15.0 ±1.1 13.3–16.5 100 ±N/A N/A 76 ±4 69–82 91 ±4 83–95 LEmin+1 mM 15.1 ±1.2 13.7–16.8 101 ±4 95–106 76 ±4 70–85 86 ±3* 80–90 FBLC2.5 mM 15.9 ±1.5* 14.0–18.1 106 ±6* 94–114 80 ±5* 73–88 90 ±3 84–94 LEmin+1.5 mM 15.9 ±1.4* 13.0–18.1 106 ±6* 94–113 81 ±5* 70–89 90 ±3 86–93 FBLC3 mM 16.8 ±1.5** 15.0–19.1 112 ±6** 102–120 85 ±5** 78–93 92 ±3 87–97 LEmin, minimum lactate equivalent; LT1, the highest stage above which blood lactate concentration increased by ≥0.1 mmol·L−1in the following stage and ≥0.2 mmol·L−1in the subsequent stage; LT0.2 mM, the stage prior to a ≥0.2 mmol·L−1blood lactate concentration elevation above baseline values; Dmax , Maximal-Deviation method; FBLC2mM, fixed blood lactate concentration (FBLC) threshold of 2 mmol·L−1; LEmin+1mM, LEmin plus 1 mmol·L−1; FBLC2.5 mM, FBLC threshold of 2.5 mmol·L−1; LEmin+1.5 mM, LEmin plus 1.5 mmol·L−1; FBLC3mM, FBLC threshold of 3 mmol·L−1. *Significantly different from MLSS (P <0.01). **Significantly different from MLSS (P <0.001). VMLSS+0.2, and did not terminate the trial. BLC during the CVRT at VMLSS+0.2 increased >1 mmol·L−1from the min 10 to the end of the trial (1.6 ±0.7 mmol·L−1;P<0.001; 90% CI: 1.26–1.95; ES: 0.82). During the CVRT at VMLSS, BLC from the 10th min to the end of the exercise increased significantly (0.4 ±0.4 mmol·L−1;P=0.02; 90% CI: 0.20–0.63; ES: 0.31), but the increment was <1 mmol·L−1in every single case. This BLC stability criterion was obtained at ≈3.9 ±1.3 mmol·L−1 (range ≈2.1–6.2). HR increased (P<0.01) over the course of both VMLSS and VMLSS+0.2 CVRTs. During the VMLSS CVRT, absolute HR increased 7 ±4 b·min−1(P<0.001; 90% CI: 5.0– 9.0; ES: 0.70) from min 10 to the end of the test. HR (%HRmax) at min 5, 10, 21 and 32 of the VMLSS CVRT were 85 ±3, 88 ±3, 91 ±4 and 92 ±4%, respectively. Correlations and Agreement Between the Measured Performance Variables Every bloody and bloodless measured endurance performance variables correlated significantly with VMLSS (Table 3). VLEmin Frontiers in Physiology | www.frontiersin.org 6July 2018 | Volume 9 | Article 1034 Garcia-Tabar and Gorostiaga Lactate Equivalent (LE) vs. MLSS TABLE 2 | Maximal values attained during the maximal ramp incremental running test (n=13). Mean ±SD Range Test duration (min:s) 12 :36 ±00 :54 11:18–14:12 PTV (km·h−1) 19.8 ±0.9 18.6–21.4 ˙ VOmax(ml·kg−1·min−1) 67.6 ±4.1 61.8–73.7 ˙ VEmax (L·min−1) 128 ±12 104–144 HRmax (b·min−1) 184 ±9 167–199 HRmax (% age predicted HRmax) 95 ±5 88–101 RERmax 1.18 ±0.05 1.09–1.26 BLCpeak (mmol·L−1) 7.6 ±2.0 5.6–11.8 PTV, peak treadmill velocity; ˙ VOmax , maximal oxygen uptake; ˙ VEmax , maximal minute ventilation; HRmax, maximal heart rate; RERmax , maximal respiratory exchange ratio; BLCpeak, peak blood lactate concentration. was the best predictor of VMLSS (Figure 4), followed by VLEmin+1 mM (r=0.86; P<0.001; SEE =0.58; 95% CI: 0.50– 1.13) and VLEmin+1.5 mM (r=0.84; P<0.001; SEE =0.86; 95% CI: 0.58–1.57). There was no statistical difference between VMLSS and estimated VMLSS using the formula exposed in Figure 4 (P=0.99; 90% CI: −0.22 to 0.22; ES: 0.001). Mean bias and LOAs were 0.00 ±0.45 km·h−1and ±0.89 km·h−1, respectively, indicating that prediction of VMLSS from VLEmin could be biased up to 5.9% above or below actual VMLSS. VLEmin+1 mM did not differ from VMLSS (P=0.47; 90% CI: −0.17 to 0.42; ES: 0.09). Mean difference was −0.12 ±0.6 km·h−1 and LOAs were ±1.18 km·h−1(±7.8%). Very large associations between VLEmin and VMLSS in absolute values (km·h−1) with their respective velocities in relative values (%PTV) were observed (Figure 5). According to the ANCOVA results, the slopes of these both regression lines were not different (P>0.05). Very large associations were also found between HR at LEmin (HRLEmin) and HR throughout the VMLSS CVRT. These correlation magnitudes were r=0.90 (Figure 6), r=0.85 (P<0.001; SEE =4.9; 95% CI: 0.30–0.83), r=0.79 (P=0.004; SEE =6.6; 95% CI: 0.25–0.96) and r=0.74 (P=0.009; SEE =7.9; 95% CI: 0.20–1.06) for HR at min 5, 10, 21, and 32 of the VMLSS CVRT, respectively. Due to some technical problems with the HR monitors, HR linear regressions are based upon 11 data-points. DISCUSSION The major finding of this study was that VLEmin was the strongest predictor of VMLSS, followed by VLEmin+1 mM,VLEmin+1.5 mM, LT1and the rest of the predictor variables (Table 3). These findings are in line with previous research showing that BLRTs, such as OBLA (Beneke, 1995; Van Schuylenbergh et al., 2004; Denadai et al., 2005; Vobejda et al., 2006; Figueira et al., 2008; Grossl et al., 2012), IAT (Beneke, 1995), Dmax (Van Schuylenbergh et al., 2004), LT (Philp et al., 2008), or other BLRTs (Grossl et al., 2012) obtained during an incremental singletest are significant determinants of MLSS. The high sustained variance by VLEmin in VMLSS prediction in this study (83%, Figure 4) is among the highest reported in the literature (50– 88%). Homogeneity of the sample, specificity and characteristics of the test protocol, precision and stability criterion in MLSS determination, as well as the exact variables derived from the incremental test chosen for BLRTs determination are potential factors affecting correlation magnitude differences among studies. For instance, endurance trained runners in the present study were relatively homogeneous in terms of VMLSS (CV ≈7%), and determination of their MLSS was very accurate (±0.2 km·h−1;±1.3% mean VMLSS). In contrast, study samples in the above-cited publications were more heterogeneous (CVs 7–16%) and precision in MLSS determination was much lower (7–15%), which are factors that can bias comparisons between studies. Concerning the aforementioned studies carried out in runners, LT (Philp et al., 2008) and OBLA (Vobejda et al., 2006) highly correlated with VMLSS, accounting for 72 and 81% of the variance, respectively. However, accuracy in VMLSS determination (3–4% mean VMLSS) was lower than in our study and the study samples composed of male and female runners were heterogeneous (VMLSS CVs 12–16%). It is well established that heterogeneity of the samples causes overestimation of correlation magnitudes; the greater the range or the heterogeneity of a group, the greater the magnitude of the correlation coefficient. With regard to prediction accuracy, it is worth mentioning the relatively low SEE (0.47 km·h−1; 3.1% mean VMLSS) in VMLSS prediction from VLEmin found in this study (Figure 4). This SEE is lower than the accuracy in MLSS identification commonly utilized (as discussed in the previous paragraph) and compares favorably with other studies predicting MLSS from the intensity associated with OBLA, where SEE values of ≈5.5% (Vobejda et al., 2006; Figueira et al., 2008) and 20.7% (Figueira et al., 2008) of mean MLSS were reported for running and cycling exercise modes, respectively. The Bland-Altman’s LOAs (±0.89 km·h−1; i.e., ±5.9% mean VMLSS) are also narrower compared to those of other studies predicting MLSS from LMT (±6.6% mean MLSS) (Sotero et al., 2009), OBLA (±10.3%) (Grossl et al., 2012) or other BLRTs (±9.5–16.5%) (Grossl et al., 2012). The strength of the relationship and prediction accuracy reported in the current study support LEmin to provide a better MLSS estimation than other BLRTs. This suggests VLEmin, an objective submaximal variable calculated during a SD-IRT, to be one of the best single VMLSS predictor in endurance trained runners. The Minimum Lactate Equivalent (LEmin) concept was first described in the 1980s by German authors (Berg et al., 1980, 1990; Lehmann et al., 1983) and was suggested to objectively represent one of the two mentioned gold standard BLC thresholds, the exercise intensity level associated with the beginning of BLC accumulation above resting values during graded exercise, nowadays known as Lactate Threshold (LT). LEmin was defined as the workload corresponding to the nadir on the quadratic relationship between BLC/workload (or ˙ VO2) ratio vs. workload (or ˙ VO2) plot-data derived from an SD-IRT. Plotting BLC/workload vs. workload turns the BLC-shape during incremental exercise into a clear “U”-BLC-shape allowing the observation of BLC/workload decrement to a nadir (LEmin) just before a clear BLC/workload increment (Figure 1). In the present Frontiers in Physiology | www.frontiersin.org 7July 2018 | Volume 9 | Article 1034 Garcia-Tabar and Gorostiaga Lactate Equivalent (LE) vs. MLSS FIGURE 3 | Mean (SD) blood lactate (triangles) and heart rate (circles) responses to the constant running velocities tests (CVRTs) at the maximal lactate steady state velocity (open symbols) and at 0.2 km·h−1faster velocity (filled symbols). * Significantly different from the rest of the time-points within the same CVRT (P<0.0125). #Significantly higher in comparison with the corresponding time-points at the maximal lactate steady state velocity CVRT (P<0.0125). study average VLEmin (11.6 km·h−1) approximate average VLT1 (11.7 km·h−1) (Table 1). This suggests LEmin to represent the pivotal equilibrium point between blood lactate production and removal (Lehmann et al., 1983; Aunola and Rusko, 1988; Berg et al., 1990). LEmin might be associated with several physiological characteristics and mechanisms, such as glycolytic acceleration, muscle oxidative capacity, type II muscle fiber recruitment, intramuscular lactate production, lactate release and clearance, capillary density and increasing concentrations of circulating hormones (Ivy et al., 1980; Lehmann et al., 1983; Gladden, 2004). The reason why LEmin would offer significant prediction advantages over other BLRTs to estimate VMLSS can be related to: (1) the resolution of LEmin determination is finer than other BLRTs (e.g., LT) because all the data points before and after the transition are used to project the LEmin value; (2) undesired error effects due to statistical scatter of the data points are minimized by the least squares curve-fitting procedure; (3) LEmin could essentially take on an infinite number of values, whereas LT1 and LT0.2 mM could only be based on the discrete values of the specific velocity-rate stages; (4) the troublesome identification of the first BLC elevation above baseline values (LT) due to initial BLC fluctuations associated with the error of the analyzer (Weltman et al., 1987) is resolved by the “U”-BLC-shape of LEmin identification without the need of a previous high level of exertion phase to induce hyperlactatemia, as it is required for LTM identification; and (5) relative changes in BLC based on the shape and slope of the BLC/workload vs. workload curve (i.e., LEmin) during incremental exercise may be more advantageous, sensitive and robust compared with the use of absolute BLC values (i.e., FBLC thresholds) (Dickhuth et al., 1999). The relevance of LEmin as a predictor variable is underpinned by the fact that the other two LEmin-related thresholds (LEmin+1 mM and LEmin+1.5 mM) were the second and third variables best correlated with VMLSS. Additionally, LEmin, LEmin+1 mM and LEmin+1.5 mM were the best ˙ VO2max predictors, whereas average VLEmin+1 mM (15.1 km·h−1) was nearly identical to average VMLSS (15.0 km·h−1). This suggests that VLEmin+1 mM may provide a close approximation of VMLSS.These results, therefore, support LE/running-velocity to be a very good predictor of the individual and group average VMLSS in endurance trained runners. A substantial relationship (r=0.85) was observed between VMLSS and %PTV at VMLSS. A similar correlation magnitude (r=0.83) was observed between VLEmin and %PTV at VLEmin. According to our previous observations (Garcia-Tabar et al., 2015b; Llodio et al., 2015, 2016) and others (Hurley et al., 1984), these associations denote that those runners with higher VLEmin and VMLSS are more likely to possess their VLEmin and VMLSS at a higher %PTV (or % ˙ VO2max) compared to those runners with lower aerobic conditioning. It also indicates that %PTV and %˙ VO2max do not adequately differentiate across subjects, and subsequently, that the relative PTV/ ˙ VO2max concept for training prescription purposes should be used with cautious (GarciaTabar et al., 2017). Prescribed training by relative PTV/ ˙ VO2max induces different training adaptation responses (Buchheit et al., 2010), most probably due to the differed level of metabolic acidosis across individuals at a given %PTV or % ˙ VO2max (Katch et al., 1978; Meyer et al., 1999), as Figure 5 depicts. One interesting additional finding was that VLEmin to VMLSS ratio was remarkably homogeneous among subjects (77% VMLSS, range: 74–80%) in comparison with the rest of the BLRTs exposed Frontiers in Physiology | www.frontiersin.org 8July 2018 | Volume 9 | Article 1034 Garcia-Tabar and Gorostiaga Lactate Equivalent (LE) vs. MLSS TABLE 3 | Pearson’s correlation magnitudes between the selected endurance performance variables (n=13). LEmin LEmin + 1 mM LEmin + 1.5 mM LT1Dmax FBLC3 mM V90 FBLC2.5 mM PTV FBLC2 mM LT0.2 mM ˙ VO2max MLSS LEmin 0.947*** 0.928*** 0.750** 0.756** 0.813*** 0.746** 0.778** 0.718** 0.692** 0.674** 0.724** 0.912*** LEmin+1 mM 0.900*** 0.684** 0.719** 0.817*** 0.783** 0.768** 0.674* 0.665* 0.713** 0.720** 0.863*** LEmin+1.5 mM 0.703** 0.779** 0.836*** 0.596* 0.793*** 0.771** 0.706** 0.641* 0.790*** 0.839*** LT10.821*** 0.910*** 0.860*** 0.924*** 0.770** 0.920*** 0.744** 0.528 0.836*** Dmax 0.895*** 0.891*** 0.909*** 0.743** 0.902*** 0.750** 0.374 0.827*** FBLC3 mM 0.850*** 0.995*** 0.793*** 0.967*** 0.773** 0.582* 0.804*** V90 0.872*** 0.669* 0.889*** 0.864*** 0.360 0.799** FBLC2.5 mM 0.793*** 0.987*** 0.773** 0.526 0.792*** PTV 0.770** 0.777** 0.703** 0.760** FBLC2 mM 0.745** 0.422 0.734** LT0.2 mM 0.544 0.716** ˙ VO2max 0.597* LEmin, minimum lactate equivalent; LEmin+1mM, LEmin plus 1 mmol·L−1; LEmin+1.5 mM, LEmin plus 1.5 mmol·L−1; LT1, the highest stage above which blood lactate concentration increased by ≥0.1 mmol·L−1in the following stage and ≥0.2 mmol·L−1in the subsequent stage; Dmax, Maximal-Deviation method; FBLC3mM, fixed blood lactate concentration (FBLC) threshold of 3 mmol·L−1; V90, velocity corresponding to 90% of maximal heart rate; FBLC2.5 mM, FBLC threshold of 2.5 mmol·L−1; PTV, peak treadmill velocity; FBLC2mM, FBLC threshold of 2 mmol·L−1; LT0.2 mM, the stage prior to a ≥0.2 mmol·L−1blood lactate concentration elevation above baseline values; ˙ VOmax , maximal oxygen uptake. *P<0.05, **P<0.01, ***P<0.001. in Table 1 (e.g., 64–88% and 73–94% for LT1and LT0.2 mM, respectively). This low range (±3%) of the percentage of VMLSS at which vLEmin occurs is very close to the limit of the test-retest variability of MLSS workload determination (Hauser et al., 2013). This indicates that the VLEmin to VMLSS ratio is independent of the endurance capacity level of the assessed runners. Although VMLSS is substantially higher than VLEmin, it is likely that some degree of commonality exists among these two physiological parameters suggesting VLEmin as a major VMLSS determinant. Our study advocates factors controlling VLEmin to be shared, at least partly, with those controlling VMLSS. Concerning our secondary purpose, V90 was the best bloodless predictor of VMLSS, accounting for 64% of the variance, followed by PTV (58%) and ˙ VO2max (36%) (Table 3). The magnitude of the relationship between V90 and VMLSS was similar to that between FBLC thresholds and VMLSS. In addition, V90 was a strong (r=0.85–0.89) predictor of FBLC thresholds. These findings are in close agreement with previous HR-based studies in professional team-sport players (Garcia-Tabar et al., 2015b), elite Basque-ball players (Garcia-Tabar et al., 2017) and low-level (VMLSS ≈13.6 km·h−1) endurance runners (Kuphal et al., 2004) in which V90 was largely associated with VMLSS (Kuphal et al., 2004) and FBLC thresholds (Garcia-Tabar et al., 2015b, 2017). The relevance of V90 as a bloodless predictor of BLRTs is strengthened by (1) the relationship between V90 and BLRTs is quite stable despite alterations in BLRTs due to training, detraining or hypoxia (Hurley et al., 1984; Foster et al., 1999; Friedmann et al., 2004); (2) increases in V90 have been verified to predict longitudinal training-induced improvements in FBLC thresholds (Garcia-Tabar et al., 2017); and (3) V90 is determinable during a submaximal test, i.e., maximal exertion is not always necessary (Garcia-Tabar et al., 2017), what makes V90 sometimes more suitable than PTV and ˙ VO2max. Results indicate V90 to be an appealing variable since it is a valid, easy, noninvasive and low-cost suitable estimator of VMLSS and FBLC thresholds during a progressive running test in endurance trained runners facilitating the monitoring of aerobic conditioning. During exercise at VMLSS, absolute HR markedly differed between subjects. Average relative HR (%HRmax), instead, was maintained within a reasonably narrow range over time (85– 92% from min 5 to 30), although, in agreement with other studies in runners (Haverty et al., 1988; Llodio et al., 2016), it also significantly increased over time (Figure 3). This suggests that a HR zone, rather than a fixed absolute or relative HR value, should be considered during training sessions when the goal is to reach an exercise intensity related to VMLSS. However, the individual %HRmax values during VMLSS CVRTs varied considerably between individuals, ranging from 81 to 85% HRmax and from 85 to 98% HRmax after 5 and 30 min of exercise, respectively. This indicates that the HR zone corresponding to MLSS should be estimated on individual basis (Llodio et al., 2016). An interesting finding was the extremely large relationship observed between the individual absolute HRLEmin and the individual absolute HR values after 5 min at VMLSS (Figure 6). This association suggests that HRLEmin can be accurately used to predict HR value after 5 min at VMLSS. Determination of VLEmin and its corresponding HR is therefore Frontiers in Physiology | www.frontiersin.org 9July 2018 | Volume 9 | Article 1034