RESEARCH ARTICLE
Advances of the reverse lactate threshold test:
Non-invasive proposal based on heart rate
and effect of previous cycling experience
Leonardo Henrique Dalcheco Messias☯, Emanuel Elias Camolese Polisel☯, Fu´lvia
Barros Manchado-Gobatto*☯
School of Applied Sciences, University of Campinas, Limeira, Sao Paulo, Brazil
☯ These authors contributed equally to this work.
* fulvia.gobatto@fca.unicamp.br
Abstract
Our first aim was to compare the anaerobic threshold (AnT) determined by the incremental
protocol with the reverse lactate threshold test (RLT), investigating the previous cycling
experience effect. Secondarily, an alternative RLT application based on heart rate was pro-
posed. Two groups (12 per group-according to cycling experience) were evaluated on cycle
ergometer. The incremental protocol started at 25 W with increments of 25 W at each 3 min-
utes, and the AnT was calculated by bissegmentation, onset of blood lactate concentration
and maximal deviation methods. The RLT was applied in two phases: a) lactate priming seg-
ment; and b) reverse segment; the AnT (AnTRLT) was calculated based on a second order
polynomial function. The AnT from the RLT was calculated based on the heart rate (AnTRLT-
HR) by the second order polynomial function. In regard of the Study 1, most of statistical pro-
cedures converged for similarity between the AnT determined from the bissegmentation
method and AnTRLT. For 83% of non-experienced and 75% of experienced subjects the
bias was 4% and 2%, respectively. In Study 2, no difference was found between the AnTRLT
and AnTRLT-HR. For 83% of non-experienced and 91% of experienced subjects, the bias
between AnTRLT and AnTRLT-HR was similar (i.e. 6%). In summary, the AnT determined by
the incremental protocol and RLT are consistent. The AnT can be determined during the
RLT via heart rate, improving its applicability. However, future studies are required to
improve the agreement between variables.
Introduction
Over the last fifty years, the anaerobic threshold concept (AnT) has been used inside the sports
sciences and clinical contexts. This concept was initially proposed for cardiac patients [1], but
its application is also considered for control and prescription of individualized exercise inten-
sity in trained individuals [2]. Physiological parameters such as respiratory and metabolic are
commonly used to identify the AnT [3]. In this sense, the Maximal Lactate Steady State proto-
col (MLSS) [4, 5] is considered the gold standard protocol for this determination. The MLSS
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OPEN ACCESS
Citation: Messias LHD, Polisel EEC, Manchado-
Gobatto FB (2018) Advances of the reverse lactate
threshold test: Non-invasive proposal based on
heart rate and effect of previous cycling experience.
PLoS ONE 13(3): e0194313. https://doi.org/
10.1371/journal.pone.0194313
Editor: Pedro Tauler, Universitat de les Illes
Balears, SPAIN
Received: May 30, 2017
Accepted: February 28, 2018
Published: March 13, 2018
Copyright: © 2018 Messias 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 work was supported by Conselho
Nacional de Desenvolvimento Cientı´fico e
Tecnolo´gico (Award Number:302827/2015-3;
Recipient: Fu´lvia de Barros Manchado-Gobatto)
and Fundac¸ão de Amparo à Pesquisa do Estado de
São Paulo (Award Number:2012/06355-2;
Recipient: Fu´lvia de Barros Manchado-Gobatto).
The funders had no role in study design, data
has been characterized as the highest intensity that may be supported over time without blood
lactate accumulation [5]. However, the necessity of several evaluation days hinders the MLSS
application.
Conversely, single-session tests such as the incremental protocol (i.e. graded exercise test)
are valid alternatives for the AnT identification. During this application, the elevation of the
intensity increases the pyruvate rate production that eventually exceeds the maximal rate of
pyruvate oxidation, resulting in the formation of lactate. Thus, the AnT is identified during an
incremental protocol based on the non-linear increase or a distinct change in the inclination
of blood lactate curve [6, 7]. Although mathematical procedures like bissegmentation of two
linear regressions [8, 9], fixed blood lactate concentration [10] or maximal perpendicular dis-
tance [11] were proposed and extensively used to overcome disadvantages of the visual identi-
fication [12], criticism still persists regarding the AnT determination during the incremental
protocol [13, 14].
Based on such criticisms, Dotan [15] proposed a novel-testing coined as Reverse Lactate
Threshold test (RLT). This application is conducted in two complementary phases. During the
lactate priming segment phase (phase 1) individuals are submitted to a controlled warm-up
(i.e. 2–3 stages) followed by two subsequent stages at and above the predicted AnT intensity,
respectively. Thereafter, during the reverse segment phase (phase 2) the intensity is stepped
down in the subsequent stages. Once intensity declines below the AnT, the blood lactate con-
centration falls until the appearance-disappearance balance is attained at the highest point of
the reverse plot between intensity vs blood lactate concentration. According to the RLT propo-
nent, this point denotes the AnT intensity. Apart from the interesting application, this original
study was underpowered by the reduced sample evaluated.
Regarding the comparison between the incremental protocol and the RLT, Wahl et al., [16]
showed (among other results) high accuracy between these applications to determine the
MLSS. However, these authors proposed a modified RLT protocol, and the replication of the
Dotan [15] findings is still required, mainly considering a larger sample. Additionally, despite
heart rate is a valuable tool to link the laboratory results to field conditions [17, 18], the effect
of the heart rate for AnT identification via the RLT was not tested. Due to its non-invasive
nature, this analysis represents a valuable tool for athletes and non-athletes. During an incre-
mental protocol blood lactate concentration commonly present a curvilinear kinetic [19] while
heart rate increases linearly or often presents an exponential decrement [20]. However, during
the reverse segment of the RLT, the heart rate presents a smoothed fashion [15] that theoreti-
cally enables the identification of AnT by the heart rate. Similar procedure was already con-
ducted for healthy [21] and disabled [22] individuals in the lactate minimum test [23], which
in turn shares some similarity within the RLT protocol. In order to increase the RLT applica-
bility, we investigated the AnT identification during this protocol using the heart rate.
Therefore, the first aim of this study was to compare in cycling exercise the AnT deter-
mined by distinct mathematical models on traditional incremental protocol with those from
the RLT. Secondarily, we aimed to test an alternative and non-invasive proposal for the AnT
determination in terms of heart rate during the RLT. Moreover, both aims were tested consid-
ering the effect of the previous experience in cycling exercise.
Materials and methods
Subjects were asked to keep the same individual hydration/food habits and avoid hard physical
activity, alcohol and caffeine ingestion at least 96 hours prior the tests. Sample size estimation
was performed using the G-Power software [24], considering 1-β = 0.90 and α = 0.05. Accord-
ing to it, ten subjects per group were enough for the aims of this study. Therefore, twenty-four
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collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
active, non-athletes, and non-smokers men were divided in two groups (12 per group). Group 1
consisted of individuals (age = 24±2 years, body mass = 78.1±11.9 kg, height = 178±1 cm, body
fat = 8.1±5.3%) that have a minimum of two years’ experience (3 times a week for at least 60
minutes) in cycling exercise, including mountain bike and speed subcategories. On the other
hand, in Group 2 were included individuals (age = 23±3 years, body mass = 67.9±7.8 kg,
height = 173±1 cm, body fat = 7.9±4.3%) that perform (3 times a week for at least 60 minutes)
distinct activities (e.g. resisted training, soccer, basketball, etc.), but did not have any experience
in cycling exercise. The level of physical activity was analyzed by the International Physical Activ-
ity Questionnaire (IPAQ) (Group 1 = 4985±3196 MET-min/wk; Group 2 = 3218±1803 MET-
min/wk). Participants provided written, informed consent authorizing their participation in this
study. All experiments were approved by the Faculty of Medical Sciences Ethics Committee (n˚
41257314.5.0000.5404) and were conducted according to the ethical international standards.
Design
Twenty four subjects completed three visits to the laboratory, 36–48 hr apart. The first visit
was conducted as a familiarization session (i.e. 30 minutes of cycling at fixed 80 rpm) and also
for the anthropometric evaluation. During the second session, participants underwent an
incremental exercise testing for AnT determination. Such procedure was necessary for the
third evaluation session, in which the RLT protocol was carried with basis on the AnT. Regard-
ing our first aim, we compared the AnT from a traditional incremental protocol with those
from the RLT, considering the previous cycling experience effect. In light of our positive
results, we tested the possibility to determine non-invasively the AnT from the RLT protocol
in terms of heart rate. The previous experience effect was also considered in such perspective.
The approaches from Cohen, Bland and Altman, Pearson and Fisher were used for analysis of
magnitude of differences, agreement, relationship and comparison of variances [25–27].
Procedures
All procedures were conducted in a controlled environment throughout experiment
(temperature = 22˚C±1˚C; relative humidity = 50%±2%; luminosity = ~300lx. The protocols
were carried on a Monark cycle ergometer (Monark Ergomedic 894 E, Monark, Sweden) at
the same time of day. While experienced cyclists (Group 1) prefer high cadences (>85 rpm),
non-experienced (Group 2) have opposite preferences (<75 rpm) [28]. However, evidences
conclude the delta efficiency (i.e. indirect measurement of muscular efficiency) seems to be
not affected by the cycling experience [28]; therefore, we opted for the 80 rpm, since it is an
intermediary cadence between experienced and non-experienced individuals. The power was
directly analyzed via a module USB 6008 (National Instruments, TX, US) connected to the
cycle ergometer. Throughout testing, signals were collected at 1000 Hz frequency. Thereafter,
were processed and analyzed using LabView-Signal-Express 2.0 (National Instruments, TX,
US) and Matlab (Mathworks, MA, US), respectively. The power was calculated as: Power =
(RF (EDT x PR))/6.12, where RF = resistance on the flywheel (kg); EDT = effective distance
travelled (m), which was equal to 6.03 (i.e. circumference of the flywheel resistance track—
1.625 m times the revolutions on the flywheel—3.714 resulted from one complete revolution);
PR = pedal revolution; 6.12 is the constant for the conversion of kpm to watts [29].
Aim 1 –Comparison between the incremental protocol and RLT results in
experienced and non-experienced subjects
The incremental protocol started at initial power output of 25 W, with increments of 25 W at
every 3 minutes. The exhaustion criteria were considered as non-maintenance of the predicted
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cadence, attainment of the predicted maximum heart rate ((210)—age0,65) [30] or volitional
exhaustion. Subjects were instructed to maintain the 80 rpm cadence throughout the test. At
the end of each stage, capillarized blood samples (25 μl) were taken from the earlobe and
deposited into microtubes (Eppendorf 1.5 ml) containing 50 μl of NaF. The blood L-lactate
concentration was analyzed by the electrochemical method using a lactimeter YSI-
2300-STAT-Plus (Yellow Springs, OH, USA).
Three mathematical models were used to calculate the AnT and blood lactate/heart rate at
the AnT. Regarding the bissegmentation method (i.e. two-segment linear fit) [8, 9], blood lac-
tate concentration was plotted against power and the lactate breakpoint was visually identified
by three independent and experienced researchers; subsequently, the AnT (AnTInc-Biss) was
determined by the intersection of the two linear fit. The blood lactate concentration at anaero-
bic threshold intensity ([Lac]AnT-Inc-Biss) was calculated by linear interpolation. The mean
heart rate of each stage was plotted against power and the heart rate at anaerobic threshold
intensity (HRAnT-Inc-Biss) was calculated by linear interpolation.
For obtainment of AnT and heart rate at AnT by the onset of blood lactate concentration
(OBLA) [10], the fixed blood lactate concentration at 4 (AnTInc-OBLA4 and HRAnT-Inc-OBLA4)
and 3 mmol.L-1 (AnTInc-OBLA3 and HRAnT-Inc-OBLA3) were calculated by an exponential inter-
polation on the blood lactate concentration and power curve. The blood lactate concentration
at these intensities was not calculated in this method since it uses fixed blood lactate concentra-
tion. The method defined as the maximal distance between two end points of the blood lactate
concentration vs power curve (Dmax) [11] was also conducted. Therefore, the AnT was calcu-
lated as the maximal perpendicular distance between a linear regression (considering the two
end points) and an exponential (AnTInc-DmaxExp) or 2˚ order polynomial (AnTInc-DmaxPoli)
adjustments. The blood lactate concentration at these intensities were calculated by exponen-
tial ([Lac]Inc-DmaxExp) or polynomial ([Lac]Inc-DmaxPoli) interpolation.
The time to exhaustion (Tex) was considered as the total time of exercise performed during
the incremental protocol. Heart rate was monitored using a validated monitor [31] (Polar,
RS800, RJ, BR; accuracy ± 1%). As previously described for HRAnT-Inc-Biss analysis, the mean
heart rate of each stage was plotted against power, and the HRAnT-Inc-OBLA4, HRAnT-Incremental-
OBLA3, HRInc-DmaxExp and HRInc-DmaxPoli were calculated by linear interpolation. In addition,
the maximal heart rate was obtained as the highest heart rate registered during the test
(HRmax-calc) and by predicted equation (HRmax-predic) (HRmax-predic = 210—age x 0.65) [30].
The percent of maximal heart rate at the thresholds was also calculated considering the
HRmax (%HRmax-calc) and by the predicted maximum heart rate (%HRmax-predic). Pulse oxim-
etry (SpO2) was also monitored during the test (OXIFAST, Takaoka, SP, BR).
The RLT protocol was applied according to the original study [15]. The only difference con-
sisted on the stages duration. Dotan [15] opted by the 4-min duration to avoid conflicting con-
siderations that shorter on longer durations may result. Since previous reports demonstrated
no difference in terms of AnT determination in graded exercise tests [3] within stages of
3-min or 4-min, we opted for the former to avoid lengthen evaluations. The RLT was con-
ducted with basis on the AnTInc. Capillarized blood samples were collected at the end of each
stage and blood lactate concentration was analyzed as earlier described. Heart rate and pulse
oximetry were also monitored. The RLT was applied in 27 minutes of cycling exercise in two
phases: a) Lactate-priming segment; and b) Reverse Segment (Fig 1).
During the lactate-priming segment three graded stages below the AnTInc were conducted
as a controlled warm-up. For instance, whether a subject achieved an AnTInc of 150 W, we
considered the intensities of 75, 100, 125 W during the first part of the RLT lactate-priming
segment (totalizing 9 minutes of warm-up). Subsequently, individuals performed three
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minutes in its AnT intensity and right after another stage at 20% above the AnTInc. In sum-
mary, the lactate-priming segment consisted in 15 minutes of cycling.
The reverse segment was conducted following the lactate-priming segment. In this phase,
intensity is retrograde. Decrements of 10 W (8%) were applied at each stage, totaling 12 min-
utes of cycling exercise. Considering the blood lactate concentration is elevated at the last part
of the lactate-priming segment (i.e 20% above AnTInc), it is expected that this metabolite
decays once exercise intensity was below AnTInc (i.e. during the last two stages of the reverse
segment). The reverse plot between power and blood lactate concentration was traced using a
second order polynomial function. According to the RLT proponent, the AnT from the RLT
protocol (AnTRLT) corresponds to the apex of the blood lactate concentration vs power curve.
The second order polynomial equation (y = ax2- bx + c) was considered, and the AnTRLT was
calculated as y = b / (2a), and the blood lactate concentration at AnTRLT ([Lac]AnT-RLT) as: y =
((a (AnTRLT  AnTRLT))—(b  AnTRLT) + c). The HRmax-calc, %HRmax-calc, %HRmax-predic were
also calculated. The HRmax-predic was the same for both RLT and incremental protocol, since
considers the subject’s age [30].
Aim 2 –Non-invasive determination of the AnTRLT based on heart rate in
experienced and non-experienced subjects
During the RLT the heart rate was monitored. The heart rate was plotted against power
(Fig 2) and a second order polynomial function was adopted and the apex was considered
as the AnTRLT in terms of heart rate (AnTRLT-HR). As described in the latter section, the
second order polynomial equation (y = ax2- bx + c) was considered, and the AnTRLT-HR
was calculated as y = b / (2a), and the heart rate at AnTRLT (HRAnT-RLT) as: y = ((a
(AnTRLT-HR  AnTRLT-HR))—(b  AnTRLT-HR) + c). The success rate of the AnTRLT and
AnTRLT–HR was determined considering the goodness of fit (R2) of the polynomial adjust-
ment higher than 0.90.
Fig 1. Curve of the reverse lactate threshold test considering the blood lactate concentration plotted against power from
experienced individual (subject 1 from experienced group). The second order polynomial adjustment was considered for
the anaerobic threshold determination (AnTRLT) and blood lactate concentration at anaerobic threshold ([Lac]AnT-RLT).
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Statistical analyses
Statistical procedures were conducted using a statistical software package (STATISTICA 7.0,
Statsoft, OK, USA). Mean and standard deviation (SD) were calculated for all studied variables.
Levene and Shapiro-Wilk tests confirmed the homogeneity and normality of our data. Com-
parison among the results of the incremental protocol (i.e. AnTInc, [Lac]AnT-Inc and HRAnT-Inc,
SpO2) and RLT (i.e. AnTRLT and [Lac]AnT-RLT and HRAnT-RLT, SpO2) for both experienced
and non-experienced subjects was performed by the two-way ANOVA. Similar procedures
were adopted for the comparison among the AnT determined by blood lactate concentration
and heart rate in the RLT. In the latter case, the data from the RLT in terms of blood lactate
concentration was also used in the second aim of this study. The Scheffe´ post-hoc was consid-
ered in all analysis of variance. The comparison of the Tex between experienced and non-expe-
rienced individuals was performed by a t-test for independent samples. The same test was
adopted for the comparison of the anthropometric characteristics. The agreement between
variables was analyzed by the Bland-Altman procedure [25] considering α = 0.05. Pearson
product moment (r) coefficient of variation (CV) [27] and effect sizes (ES) [26] were also cal-
culated. Cohen’s categories used to evaluate the magnitude of the ES were: small if 0  |d|
0.5; medium if 0.5 < |d|  0.8; and large if |d| > 0.8). In all cases, statistical significance was set
at P<0.05.
Results
Aim 1 –Comparison between the incremental protocol and RLT results in
experienced and non-experienced subjects
Statistical difference between groups regarding anthropometric characteristics was only visual-
ized for body mass (age-P = 0.251; body mass-P = 0.017; height-P = 0.079; body fat-P = 0.982).
ANOVA did not point difference regarding the mathematical models used for anaerobic thresh-
old intensity determination on the incremental protocol and individual’s cycling experience
Fig 2. Curve of the reverse lactate threshold test considering the heart rate plotted against power from experienced
individual (subject 1 from experienced group). The second order polynomial adjustment was considered for the
anaerobic threshold determination (AnTRLT-HR) and heart rate at anaerobic threshold (HRAnT-RLT).
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(P = 0.395). Similar results were obtained for blood lactate concentration (P = 0.818) and heart
rate (P = 0.469) at the anaerobic threshold intensity.
In regard of the comparison between incremental protocol and RLT results (Tables 1–3),
no difference was found between the anaerobic threshold intensity determined by the RLT
and the incremental protocol; such result was not affected by the previous experience in
cycling exercise (Table 1). However, only the comparison between AnTRLT and AnTInc-Biss
converge in most of statistical analysis for both non-experienced and experienced individuals.
In line with this, the agreement between these intensities were the strongest when compared
with the other mathematical methods (Fig 3A, Fig 4A).
Apart from the non-difference (for both groups) and significant relationship (only for non-
experienced subjects) between [Lac]AnT-RLT and [Lac]AnT-Inc-DmaxExp, distinct results were
found between protocols in terms of the blood lactate concentration at the AnT (Table 2).
Excepted of the significant relationship between HRAnT-RLT and HRAnT-Inc-Biss, different
results were found between protocols for heart rate at the AnT intensity (Table 3). In addition,
the Table 4 shows the results of HRmax-calc, HRmax-predic, %HRmax-calc, %HRmax-predic. The Tex
Table 1. Comparison between the anaerobic threshold intensity from the incremental protocol analyzed by different mathematical models and the reverse lactate
threshold test obtained by subjects without (non-experienced) or with (experienced) experience with cycling exercise.
AnTRLT
AnTInc-Biss
AnTInc-OBLA4
AnTInc-OBLA3
AnTInc-DmaxExp
AnTInc-DmaxPoli
(W)
(W)
(W)
(W)
(W)
(W)
Non-Experienced (n = 12)
Mean
112
108
105
87
117
95
SD
15
17
10
10
13
8
P (Scheffe´)
———
0.977
0.924
0.180
0.967
0.226
ES
———
0.24
0.60
1.86
0.34
1.42
% Diff
———
3.4
6.8
21.9
4.3
15.1
r (P)
———
0.95 (0.008)
0.50 (0.090)
0.46 (0.127)
0.81 (0.001)
0.67 (0.015)
CV
———
3.1
8.4
10.1
5.6
8.0
Bland-Altman
———
-3.9±5.0
-7.6±13.6
-24.7±14.35
4.8±9.1
-16.9±11.84
Experienced (n = 12)
Mean
155
149
167
143
164
133
SD
25
26
41
39
34
23
P (Scheffe´)
———
0.925
0.722
0.824
0.868
0.062
ES
———
0.23
0.34
0.37
0.27
0.91
% Diff
———
3.8
7.2
7.6
5.2
14.3
r (P)
———
0.98 (0.000)
0.79 (0.002)
0.75 (0.004)
0.95 (0.000)
0.87 (0.000)
CV
———
2.2
12.3
12.5
5.6
6.2
Bland-Altman
———
-6.0±4.8
11.3±26.3
-11.9±26.4
8.1±12.5
-22.3±12.6
Note: The statistical results refers to the comparison between the anaerobic threshold intensity from the reverse lactate threshold and the anaerobic threshold intensity
from the mathematical models applied in the incremental protocol. AnTRLT—Anaerobic threshold intensity determined during the reverse lactate threshold test in
terms of blood lactate concentration. AnTInc-Biss−Anaerobic threshold intensity determined during the incremental protocol by the bissegmentation method; AnTInc-
OBLA4 –Anaerobic threshold intensity determined during the incremental protocol by the fixed blood lactate concentration at 4 mmol.L-1; AnTInc-OBLA3 –Anaerobic
threshold intensity determined during the incremental protocol by the fixed blood lactate concentration at 3 mmol.L-1; AnTInc-DmaxExp−Anaerobic threshold intensity
determined during the incremental protocol by maximal deviation method using the exponential adjustment; AnTInc-DmaxPoli−Anaerobic threshold intensity
determined during the incremental protocol by maximal deviation method using the second order polynomial adjustment; P (Scheffe´)–P value from the Scheffe´ post-
hoc analysis; ES–effect size; % Diff–percent difference between means; r (P) Pearson product moment and P value from the correlation; CV–coefficient of variation;
Bland-Altman—Bland Altman analysis considering 95% of agreement limits.
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was different between non-experienced (18min 13s ± 1min 46s) and experienced (22min
54s ± 2min 48s) individuals (P = 0.000). Pulse oximetry was not different during both
incremental (non-experienced– 96.4±1.0; experienced– 96.1±1.2%; P = 0.916) and RLT
(non-experienced– 96.0±1.0; experienced– 95.4±1.2%; P = 0.695); additionally, ANOVA
also did not show interaction regarding the factorials variables (i.e. test and experience)
(P = 0.729).
Aim 2 –Non-invasive determination of the AnTRLT based on heart rate in
experienced and non-experienced subjects
The Table 5 shows the AnT determined during the RLT by means of heart rate for both
groups. All statistical procedures converged to the similarity between the results, with the
exception of the non-significant relationship (for both groups). The agreement among vari-
ables are shown on Fig 5. High R2 were obtained on the polynomial adjustment adopted for
both groups in terms of AnTRLT (Non-Experienced—range = 0.78–0.99; Experienced—
range = 0.90–0.99) and AnTRLT-HR (Non-Experienced—range = 0.90–0.99; Experienced—
Table 2. Comparison between the blood lactate concentration at the anaerobic threshold intensity from the incremental protocol analyzed by different mathemati-
cal models and the reverse lactate threshold test obtained by subjects without (non-experienced) or with (experienced) experience with cycling exercise.
[Lac]AnT-RLT
[Lac]AnT-Inc-Biss
[Lac]AnT-Inc-OBLA4
[Lac]AnT-Inc-OBLA3
[Lac]AnT-Inc-DmaxExp
[Lac]AnT-Inc-DmaxPoli
(mmol.L-1)
(mmol.L-1)
(mmol.L-1)
(mmol.L-1)
(mmol.L-1)
(mmol.L-1)
Non-Experienced (n = 12)
Mean
6.74
3.26
———
———
5.01
3.20
SD
2.63
1.29
———
———
1.03
0.86
P (Scheffe´)
———
0.013
———
———
0.417
0.009
ES
———
1.78
———
———
0.95
2.03
% Diff
———
51.6
———
———
25.6
52.5
r (P)
———
0.71 (0.008)
———
———
0.70 (0.010)
0.54 (0.065)
CV
———
27.3
———
———
24.6
32.4
Bland-Altman
———
-3.4±1.9
———
———
-1.7±2.0
-3.5±2.2
Experienced (n = 12)
Mean
6.47
2.27
———
———
4.12
1.88
SD
3.84
1.08
———
———
1.46
0.85
P (Scheffe´)
———
0.002
———
———
0.166
0.000
ES
———
1.71
———
———
0.89
1.95
% Diff
———
64.9
———
———
36.2
70.8
r (P)
———
0.29 (0.350)
———
———
0.38 (0.212)
0.07 (0.816)
CV
———
59.4
———
———
47.3
77.6
Bland-Altman
———
-4.2±3.6
———
———
-2.3±3.5
-4.5±3.9
Note: The statistical results refers to the comparison between the anaerobic threshold intensity from the reverse lactate threshold and the anaerobic threshold intensity
from the mathematical models applied in the incremental protocol. [Lac]AnT-RLT−Blood lactate concentration at the anaerobic threshold intensity determined during
the reverse lactate threshold test; [Lac]AnT-Inc-Biss−Blood lactate concentration at the anaerobic threshold intensity determined during the incremental protocol by the
bissegmentation method; [Lac]AnT-Inc-OBLA4 –Blood lactate concentration at the anaerobic threshold intensity determined during the incremental protocol by the fixed
blood lactate concentration at 4 mmol.L-1; [Lac]AnT-Inc-OBLA3 –Blood lactate concentration at the anaerobic threshold intensity determined during the incremental
protocol by the fixed blood lactate concentration at 3 mmol.L-1; [Lac]AnT-Inc-DmaxExp−Blood lactate concentration at the anaerobic threshold intensity determined during
the incremental protocol by maximal deviation method using the exponential adjustment; [Lac]AnT-Inc-DmaxPoli−Blood lactate concentration at the anaerobic threshold
intensity determined during the incremental protocol by maximal deviation method using the second order polynomial adjustment; P (Scheffe´)–P value from the
Scheffe´ post-hoc analysis; ES–effect size; % Diff–percent difference between means; r (P) Pearson product moment and P value from the correlation; CV–coefficient of
variation; Bland-Altman—Bland Altman analysis considering 95% of agreement limits.
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range = 0.90–0.99). In line with this, high success rate were obtained for the two determina-
tions in both groups.
Discussion
The main findings of the present investigation were the AnT determined on the RLT was con-
sistent with those obtained on the incremental protocol calculate by the bissegmentation
mathematical model. In addition, our results showed that previous cycling experience did not
influence on such consistency. However, due to the different methodologies, additional
parameters like blood lactate concentration and heart rate at the anaerobic threshold intensity
are not similar. Additionally, for 83% of the evaluated non-experienced individuals the differ-
ence between the AnT determined on RLT by blood lactate concentration and heart rate was
6%. Similar difference was also obtained for 91% of the evaluated experienced individuals.
Therefore, our results suggest that AnT determination via heart rate on RLT may be consid-
ered as a non-invasive alternative. However, future studies are necessary to investigate which
factors can improve the agreement between these measures.
Table 3. Comparison between the heart rate at the anaerobic threshold intensity from the incremental protocol analyzed by different mathematical models and the
reverse lactate threshold test obtained by subjects without (non-experienced) or with (experienced) experience with cycling exercise.
HRAnT-RLT
HRAnT-Inc-Biss
HRAnT-Inc-OBLA4
HRAnT-Inc-OBLA3
HRAnT-Inc-DmaxExp
HRAnT-Inc-DmaxPoli
(bpm)
(bpm)
(bpm)
(bpm)
(bpm)
(bpm)
Non-Experienced (n = 12)
Mean
171
155
152
144
159
148
SD
13
13
15
15
14
13
P (Scheffe´)
———
0.000
0.001
0.000
0.010
0.000
ES
———
1.21
1.30
1.89
0.89
1.72
% Diff
———
9.4
10.9
15.8
7.2
13.5
r (P)
———
0.64 (0.022)
0.42 (0.165)
0.35 (0.255)
0.45 (0.138)
0.37 (0.226)
CV
———
4.8
3.5
7.4
6.2
6.6
Bland-Altman
———
-16.1±11.1
-18.7±15.4
-27.1±16.4
-12.3±14.5
-23.1±15.0
Experienced (n = 12)
Mean
177
159
165
155
163
150
SD
12
10
14
13
11
8
P (Scheffe´)
———
0.007
0.321
0.007
0.088
0.000
ES
———
1.62
0.88
1.71
1.17
2.63
% Diff
———
10.4
6.6
12.3
7.8
15.2
r (P)
———
0.44 (0.146)
0.09 (0.760)
0.01 (0.959)
0.14 (0.659)
0.03 (0.907)
CV
———
5.1
4.1
7.8
6.4
6.3
Bland-Altman
———
-18.6±12.5
-11.7±19.9
-21.9±18.3
-13.8±15.5
-27.0±14.5
Note: The statistical results refers to the comparison between the anaerobic threshold intensity from the reverse lactate threshold and the anaerobic threshold intensity
from the mathematical models applied in the incremental protocol. HRAnT-RLT−Hear rate at the anaerobic threshold intensity determined during the reverse lactate
threshold test; HRAnT-Inc-Biss−Heart rate at the anaerobic threshold intensity determined during the incremental protocol by the bissegmentation method; HRAnT-Inc-
OBLA4 –Hear rate at the anaerobic threshold intensity determined during the incremental protocol by the fixed blood lactate concentration at 4 mmol.L-1; HRAnT-Inc-
OBLA3 –Hear rate at the anaerobic threshold intensity determined during the incremental protocol by the fixed blood lactate concentration at 3 mmol.L-1; HRAnT-Inc-
DmaxExp−Hear rate at the anaerobic threshold intensity determined during the incremental protocol by maximal deviation method using the exponential adjustment;
HRAnT-Inc-DmaxPoli−Hear rate at the anaerobic threshold intensity determined during the incremental protocol by maximal deviation method using the second order
polynomial adjustment; P (Scheffe´)–P value from the Scheffe´ post-hoc analysis; ES–effect size; % Diff–percent difference between means; r (P) Pearson product moment
and P value from the correlation; CV–coefficient of variation; Bland-Altman—Bland Altman analysis considering 95% of agreement limits.
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Fig 3. Bland-Altman analysis performed for anaerobic threshold intensity determined on the reverse lactate threshold test and incremental
protocol on non-experienced individuals. a) Agreement between anaerobic threshold intensity determined on incremental protocol by the
bissegmentation method (AnTInc-Biss) and reverse lactate threshold test (AnTRLT); b) Agreement between anaerobic threshold intensity determined on
incremental protocol by fixed blood lactate concentration at 4 mmol.L-1 (AnTOBLA4) and AnTRLT; c) Agreement between anaerobic threshold intensity
determined on incremental protocol by fixed blood lactate concentration at 3 mmol.L-1 (AnTOBLA3) and AnTRLT; d) Agreement between anaerobic
threshold intensity determined on incremental protocol by maximal deviation method using the exponential adjustment (AnTDmaxExp) and AnTRLT; e)
Agreement between anaerobic threshold intensity determined on incremental protocol by maximal deviation method using the second order
polynomial adjustment (AnTDmaxPoli) and AnTRLT.
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Fig 4. Bland-Altman analysis performed for anaerobic threshold intensity determined on the reverse lactate threshold test and incremental
protocol on experienced individuals. a) Agreement between anaerobic threshold intensity determined on incremental protocol by the bissegmentation
method (AnTInc-Biss) and reverse lactate threshold test (AnTRLT); b) Agreement between anaerobic threshold intensity determined on incremental
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Aim 1 –Comparison between the incremental protocol and RLT results in
experienced and non-experienced subjects
Regarding our first aim, we found no difference between the AnT determined during the RLT
and incremental protocol. On the other hand, differences in terms of blood lactate concentra-
tion and heart rate at AnT intensity were found. Despite these distinct results, the previous
experience in cycling exercise did not affect the results provided by both protocols. Overall,
both protocols seem to identify the same phenomenon (i.e. AnT) regardless of previous experi-
ence in cycling, but the physiological condition that such identification occurs depends on the
characteristic of each protocol.
The main criticism suggested by Dotan [15] regarding the incremental exercise is related to
the ambiguity of where the highest blood lactate concentration is identified. Some studies con-
verge with this approach [13, 14]. Although the RLT seems to deal with such problem, its pro-
ponent also highlights other methodological concerns [15]. For instance, the need for “on-the-
fly” blood sampling is suggested as a limited factor for sports where this is not possible (e.g.
swimming); this limitation can be extended to incremental protocols applied to these exercises.
In this sense, long and repetitive exercise breaks can lead to an AnT overestimation, since the
glycolytic rate resulted from the previous incremental stage is decreased while oxygen uptake
remains elevated, improving the blood lactate concentration clearance [32]. However, such
limitation was not present in our study. Moreover, the duration of stages were standardized
for the RLT and incremental protocol. We believe that these factors along with the RLT appli-
cation based on the incremental protocol were crucial for the similarity in terms of AnT.
Indeed, the AnT may be identified during the incremental protocol when an abrupt eleva-
tion of blood lactate concentration is visualized. In this sense, the balance of blood lactate con-
centration between lactate producing and absorbing compartments is gradually lost.
Conversely, the RLT test is conducted under an inverse concept, that is, the organism is
already facing the loss of blood lactate concentration balance between compartments (i.e. last
part of lactate-priming segment). When intensities are stepped down in the reverse segment,
the balance between blood lactate concentration production-removal is gradually recovered.
On the other hand, because at least two stages were performed above the predicted AnT (i.e.
last stage from the lactate-priming segment and first stage of the reverse segment), the blood
lactate concentration removal will be greater than its production only when the stages below
the predicted AnT were performed. Such characteristic is responsible by the high blood lactate
concentration and heart rate found in our study at the apex of the second order polynomial
adjustment, that is, at the AnTRLT (Tables 2–3).
According to Dotan [15], further experimentation is required before final validation of the
RLT. Thus, we contributed with such perspective by demonstrating that the previous cycling
experience did not affect the AnTRLT identification. It is consolidated that muscular efficiency
as well as aerobic and anaerobic power/capacity between individuals with or without cycling
experience is quite distinct [33, 34]. Despite the encouraging reports, methodological concerns
related to fitness level must be investigated. For instance, we considered the upper limit of 20%
increment proposed in the original RLT during the last stage of the lactate-priming segment.
However, since well-trained individuals present the AnT at high percentage of its maximal
protocol by fixed blood lactate concentration at 4 mmol.L-1 (AnTOBLA4) and AnTRLT; c) Agreement between anaerobic threshold intensity determined
on incremental protocol by fixed blood lactate concentration at 3 mmol.L-1 (AnTOBLA3) and AnTRLT; d) Agreement between anaerobic threshold
intensity determined on incremental protocol by maximal deviation method using the exponential adjustment (AnTDmaxExp) and AnTRLT; e)
Agreement between anaerobic threshold intensity determined on incremental protocol by maximal deviation method using the second order
polynomial adjustment (AnTDmaxPoli) and AnTRLT.
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oxygen consumption (VO2max) [35], it is possible that the 20% increment surpass its maximal
oxygen consumption intensity, leading to premature exhaustion.
Regarding the reverse segment, the individual’s physiological/metabolic condition must be
also accounted. The improvement of blood lactate clearance is considered as positive physio-
logical adaptation from endurance training [36], which can be extended to well-trained indi-
viduals. Therefore, the upper limit of 8% decrement originally proposed for the reverse
segment may lead to a faster blood lactate concentration clearance in these individuals, overes-
timating the real AnT. Regarding the stage duration, the slight modification used in this study
(i.e. 3-min instead of the original 4-min) did not implicate on the AnTRLT determination,
since high coefficient of determinations and high success rate were obtained. Other methodo-
logical concerns were considered in the study of Wahl et al., [16], in which a modified RLT
protocol was proposed to improve the test’s precision, also enabling the VO2max determination
during the lactate priming-segment. Within this new approach the RLT was more accurate to
determine the MLSS than other threshold concepts, such as OBLA4 [10] or the Dmax [11].
Overall, further studies should consider the methodological concerns highlighted along with
the results of Wahl et al., [16], mainly whether the RLT application was conducted to highly
trained individuals.
Lastly, another RLT limitation is the necessity of the previous AnT determination for inten-
sity prescription during the two phases. However, futures studies are encouraged to investigate
whether indirect methods or even the reported mean velocity during recent championships/
training sessions to estimate the intensities of the RLT. The marked differences in terms of
methodology and application implies in the use of the RLT and incremental protocol inside
Table 4. Maximal heart rate predicted (HRmax-predic) and registered (HRmax-calc) during the reverse lactate threshold test and incremental protocol, as well as the
percent of maximal heart rate at the anaerobic threshold intensities considering the registered (%HRmax-calc) and predicted (%HRmax-predic) maximal heart rate.
Non-Experienced (n = 12)
Experienced (n = 12)
RLT
HRmax-calc (bpm)
177±14
183±13
HRmax-predic (bpm)
197±3
195±2
AnTRLT
%HRmax-calc
96±1
96±1
%HRmax-predic
91±7
94±6
Incremental
HRmax-calc (bpm)
191±16
195±7
HRmax-predic (bpm)
197±3
195±2
AnTInc-Biss
%HRmax-calc
81±6
81±5
%HRmax-predic
80±7
81±5
AnTInc-OBLA4
%HRmax-calc
79±5
85±8
%HRmax-predic
78±8
85±7
AnTInc-OBLA3
%HRmax-calc
76±6
79±7
%HRmax-predic
74±8
80±7
AnTInc-DmaxExp
%HRmax-calc
83±5
83±5
%HRmax-predic
81±7
84±6
AnTInc-DmaxPoli
%HRmax-calc
77±4
77±5
%HRmax-predic
76±7
77±4
AnTRLT—Anaerobic threshold intensity determined during the reverse lactate threshold test in terms of blood lactate concentration. AnTInc-Biss−Anaerobic threshold
intensity determined during the incremental protocol by the bissegmentation method; AnTInc-OBLA4 –Anaerobic threshold intensity determined during the incremental
protocol by the fixed blood lactate concentration at 4 mmol.L-1; AnTInc-OBLA3 –Anaerobic threshold intensity determined during the incremental protocol by the fixed
blood lactate concentration at 3 mmol.L-1; AnTInc-DmaxExp−Anaerobic threshold intensity determined during the incremental protocol by maximal deviation method
using the exponential adjustment; AnTInc-DmaxPoli−Anaerobic threshold intensity determined during the incremental protocol by maximal deviation method using the
second order polynomial adjustment
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the sport and clinical contexts. For instance, evidences have been suggested that blood lactate
concentration and heart rate at the AnT are valid indicators for monitoring longitudinal train-
ing effects for different populations [17, 37]. In this sense, because during the RLT application
high values of [Lac]AnT-RLT and HRAnT-RLT are obtained, it is possible that this protocol is dis-
advantaged for utilization of these results for monitoring longitudinal training effects. This
detriment is not transposed to the incremental protocol, since the [Lac]AnT-Inc and HRAnT-Inc
are consolidated indicators for training control [17, 37]. Moreover, RLT does not requires
exhaustion, the HRmax-calc and %HRmax-calc values cannot be used in the same way as for the
incremental protocol (Table 4).
It is valid to state that the comparison among mathematical models for determining the
anaerobic threshold intensity on incremental protocol is beyond the main aim of this study.
Such results were provided to strength the comparison between results from the incremental
protocol with the reverse lactate threshold test. In line with this, the results on Table 1 showed
that only the AnTInc-Biss and the AnTRLT converged in all statistical procedures adopted. In
terms of Pearson product moment, both non-experienced and experienced were heteroge-
neous regarding AnT (non-experienced 8–15% and experienced 17–27% if considered all
mathematical models), which could have improved the correlation coefficients.
In regard of the agreement, the bias from Bland-Altman analysis revealed that in some
non-experienced subjects the AnTRLT overestimated the AnTInc-Biss in ~9W. This result was
Table 5. Comparison between the results from the reverse lactate threshold test analyzed in terms of blood lactate concentration and heart rate obtained by subjects
without (non-experienced) or with (experienced) experience with cycling exercise.
AnTRLT
AnTRLT-HR
AnTRLT
AnTRLT-HR
AnTRLT
AnTRLT-HR
(W)
(W)
R2
R2
Success rate (%)
Success rate (%)
Non-Experienced (n = 12)
83.33 (n = 10)
100 (n = 12)
Mean
112
112
0.93
0.96
SD
15
16
0.07
0.03
P (Scheffe´)
0.999
0.331
ES
0.02
0.69
% Diff
0.3
3.9
r (P)
0.95 (0.000)
0.29 (0.354)
CV
3.0
5.4
Bland-Altman
0.2±4.8
-0.0±0.0
Experienced (n = 12)
91.66 (n = 11)
100 (n = 12)
Mean
155
156
0.96
0.97
SD
25
27
0.04
0.03
P (Scheffe´)
0.999
0.998
ES
0.04
0.10
% Diff
0.6
0.3
r (P)
0.95 (0.000)
0.14 (0.656)
CV
3.8
3.8
Bland-Altman
1.0±8.4
0.0±0.0
AnTRLT—Anaerobic threshold intensity determined during the reverse lactate threshold test in terms of blood lactate concentration; AnTRLT-HR—Anaerobic threshold
intensity determined during the reverse lactate threshold test in terms of heart rate; AnTRLT R2—goodness of fit of the polynomial adjustment between power and blood
lactate concentration (reverse segment); AnTRLT-HR R2—goodness of fit of the polynomial adjustment between power and heart rate (reverse segment); Success rate—
goodness of fit of the polynomial adjustment higher than 0.90. P (Scheffe´)–P value from the Scheffe´ post-hoc analysis; ES–effect size; % Diff–percent difference between
means; r (P) Pearson product moment and P value from the correlation; CV–coefficient of variation; Bland-Altman—Bland Altman analysis considering 99% of
agreement limits.
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confirmed in subjects 3 and 10, in which the AnTRLT was 14 and 10 W (respectively) higher
than the AnTInc-Biss. However, without these subjects the bias is reduced to ~5W. Overall, for
83% of non-experienced subjects this overestimation only represents 4%. This perspective is
extended for experienced subjects. In this case the bias is ~8W, which was confirmed for sub-
jects 7, 10 and 12 (i.e. AnTRLT overestimated the AnTInc-Biss in 10, 8 and 17 W, respectively).
However, without these subjects the bias is reduced to ~4W, which represents an overestima-
tion of only 2% for 75% of experienced subjects.
Although fixed blood lactate concentration enables the AnT determination without the
exhaustion attainment in incremental protocols [10], some studies have demonstrated that
depending on the mathematical model adopted, the AnT determination can be influenced [38,
39]. Therefore, despite the excessive blood sampling may be not suitable for practical contexts,
the exhaustion attainment is interesting for the robust analysis of the AnT that is being mea-
sured. Moreover, the reliable acquisition of additional parameters (e.g. maximal oxygen con-
sumption, velocity at maximal oxygen consumption or time to exhaustion) requires that
individuals reach exhaustion. However, the exhaustion attainment necessity may be not
Fig 5. a) Agreement performed by Bland-Altman analysis for anaerobic threshold intensity determined on the reverse lactate threshold test by blood
lactate concentration (AnTRLT) and heart rate (AnTRLT-HR) for non-experienced individuals; b) Agreement performed by Bland-Altman analysis
AnTRLT and AnTRLT-HR for experienced individuals; c) Agreement performed by Bland-Altman analysis for coefficient of determination on the reverse
lactate threshold test by blood lactate concentration (AnTRLT R2) and heart rate (AnTRLT-HR R2) for non-experienced individuals; d) Agreement
performed by Bland-Altman analysis AnTRLT R2 and AnTRLT-HR R2 for experienced individuals.
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suitable for untrained individuals. In this sense, the non-exhaustive nature of the RLT is an
interesting possibility for the AnT identification in peculiar cases, such as untrained or inexpe-
rienced subjects. Therefore, although no differences between AnTInc and AnTRLT were found,
we suggest that future studies should consider its applications according to the population that
will be evaluated.
Aim 2 –Non-invasive determination of the AnTRLT based on heart rate in
experienced and non-experienced subjects
Heart rate has been considered for monitoring training since 1980s [17]. Sooner after the
expansion of heart devices, Conconi et al., [40] proposed the AnT estimation based on this
physiological parameter. Nowadays the individualized exercise prescription as well as load
controlling based on heart rate are commonly used in practice [17, 18, 41, 42], since factors as
easy applicability and reduced financial cost improve the heart rate devices utilization. Addi-
tionally, in a recent meta-analysis Bellenger et al., [41] concluded the autonomic heart rate reg-
ulation may be considered a potential indicator for overreaching status.
Since Dotan [15] demonstrated the heart rate presented a smoothed fashion during the
reverse segment, it also opened for discussion if the AnT could be determined based on this
physiological parameter. In accordance, our results showed the AnT is determined during
RLT based on of heart rate. Moreover, this determination was not affected by the previous
experience in the cycling exercise. These results improve the scientific knowledge around the
RLT application, since the heart rate is a cheap and easy non-invasive tool.
The AnT determination via heart rate during the RLT was possible for all subjects, as
exposed in Table 5. In fact, the success rate of AnT determination via blood lactate concentra-
tion for two non-experienced (subject 8 –R2 = 0.83; subject 9 –R2 = 0.78) and one experienced
(subject 11 –R2 = 0.89) was slightly lower. On the other, the success rate for the AnT via heart
rate was 100% for both non-experienced and experienced subjects. In fact, while the blood lac-
tate concentration tends to decrease significantly once the equilibrium between production-
removal is gradually recovery, the cardiovascular system tends to readjust after the stressful
stage (i.e. 20% above the AnT) in a smoothed fashion [15](Fig 2). However, such differences
did not affect the second order polynomial adjustment, since high R2 were found for all sub-
jects in the AnT determination via blood lactate concentration and heart rate.
Apart from the non-difference, low effect size and significant correlation, the comparison
between AnT determined from both blood lactate concentration and heart rate resulted in a
bias of ~8 and ~15W in the Bland-Altman analysis for non-experienced and experienced sub-
jects, respectively. As discussed in the previous section, such bias can be related with the results
of few subjects. For instance, the difference of AnTRLT and AnTRLT-HR for subjects 6 and 7 (i.e.
Group 1) was ~7 and ~8W, respectively. Without this difference, the bias is reduced to ~6W
which represents a difference of AnTRLT and AnTRLT-HR of 6% for 83% of evaluated non-expe-
rienced subjects.
Regarding the experienced individuals, the bias was ~15W. For this group, the heart rate
behavior during the RLT reverse segment for one experienced subject was markedly different
from the others (Fig 6). It is clear to notice the high decrement of heart rate mainly in the two
last stages of the test. Such behavior can be related with a low efficiency in terms of readjust-
ment of autonomic nervous system along with the sympathetic-parasympathetic regulation on
the cardiovascular system [41]. Within this result, the zero tangent in x-axis of the polynomial
adjustment was right shifted, overestimating the AnT via heart rate in 13% (180 W) when
compared to the blood lactate concentration determination (159 W). This overestimation is
also extended when considered the AnTInc-Biss of this subject (158W). Without this subject, the
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bias of AnTRLT and AnTRLT-HR for the experienced subjects is reduced for ~10W, which
means that for 91% of these individuals the difference may be 6%.
Overall, we do not believe that differences invalidate the AnT determination via heart rate
during the RLT. Every proposed test to determine the AnT has a bias. The major question in
this issue is related within the magnitude of the error. It is consolidated that heart rate is a valu-
able maker for monitoring training effects or even determine physiological phenomenon such
as the AnT [17, 18, 41]. Our results corroborate with this perspective. However, every mea-
surement must be carefully analyzed in order to identify factors that are culminating for dis-
crepancies between methods, as demonstrated on Fig 6. Therefore, the results presented in this
study strengthen the determination of AnT in the RLT via heart rate, which can a valid and
useful tool.
Conclusions
In summary, the AnT determined from the RLT is consistent with those determined from the
incremental protocol by means of the bissegmentation model. However, caution is required
regarding the comparison of other physiological results (blood lactate concentration and heart
rate at AnT), since protocols are different considering methodology, resulting in distinct
results. Additionally, the previous experience in cycling exercise did not affect the AnT from
such application, highlighting the robustness of this RLT for AnT determination. Moreover,
we demonstrated the AnT determination using heart rate is a valid non-invasive alternative,
and it’s also not a function of the previous experience in cycling exercise. However, for 83%
and 91% of the evaluated non-experienced and experienced individuals (respectively), the dif-
ference between the AnT determined on RLT by blood lactate concentration and heart rate
was 6%. Therefore, future studies are necessary to investigate which factors can improve the
agreement between these measures.
Supporting information
S1 File. Datasheet with all data used in the manuscript. Folder “IncrementalTestBiss” con-
tains the data regarding the bissegmentation method. Moreover, this folder contains general
Fig 6. a) Curve of the reverse lactate threshold test from subject 5 (Experienced group) considering the blood lactate concentration plotted against power. The second
order polynomial adjustment was considered for the anaerobic threshold determination (AnTRLT); b) Curve of the reverse lactate threshold test from subject 5
(Experienced group) considering the heart rate plotted against power. The second order polynomial adjustment was considered for the anaerobic threshold
determination (AnTRLT-HR).
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information regarding the incremental protocol, such as the calculated and predicted maximal
heart rate (HRmax-Incremental-calculated and HRmax-Incremental-predicted), time to
exhaustion (Tex) and pulse oximetry (SpO2). Folder “IncrementalTestOBLA” contains the
data regarding the fixed blood lactate concentration method considering 3 and 4 mmol.L-1.
Folder “IncrementalTestDMAX” contains the data regarding the maximal perpendicular dis-
tance method considering exponential and polynomial second order adjustments. Folder
“ReverseLactateThresholdTest”contains the data regarding the Reverse Lactate Threshold
Test.
(XLSX)
Acknowledgments
We would like to thank the subjects for the participation on the procedures.
Author Contributions
Conceptualization: Leonardo Henrique Dalcheco Messias, Emanuel Elias Camolese Polisel,
Fu´lvia Barros Manchado-Gobatto.
Data curation: Leonardo Henrique Dalcheco Messias, Emanuel Elias Camolese Polisel, Fu´lvia
Barros Manchado-Gobatto.
Formal analysis: Leonardo Henrique Dalcheco Messias, Emanuel Elias Camolese Polisel, Fu´l-
via Barros Manchado-Gobatto.
Funding acquisition: Fu´lvia Barros Manchado-Gobatto.
Investigation: Leonardo Henrique Dalcheco Messias, Emanuel Elias Camolese Polisel, Fu´lvia
Barros Manchado-Gobatto.
Methodology: Leonardo Henrique Dalcheco Messias, Emanuel Elias Camolese Polisel, Fu´lvia
Barros Manchado-Gobatto.
Project administration: Fu´lvia Barros Manchado-Gobatto.
Resources: Fu´lvia Barros Manchado-Gobatto.
Supervision: Leonardo Henrique Dalcheco Messias, Fu´lvia Barros Manchado-Gobatto.
Validation: Leonardo Henrique Dalcheco Messias, Fu´lvia Barros Manchado-Gobatto.
Visualization: Leonardo Henrique Dalcheco Messias, Fu´lvia Barros Manchado-Gobatto.
Writing – original draft: Leonardo Henrique Dalcheco Messias, Emanuel Elias Camolese
Polisel, Fu´lvia Barros Manchado-Gobatto.
Writing – review & editing: Leonardo Henrique Dalcheco Messias, Emanuel Elias Camolese
Polisel, Fu´lvia Barros Manchado-Gobatto.
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Advances of the reverse lactate threshold test: Non-invasive proposal based on heart rate and effect of previous cycling experience.
Messias, Leonardo Henrique Dalcheco,Polisel, Emanuel Elias Camolese,Manchado-Gobatto, Fúlvia Barros