RESEARCH ARTICLE
Manipulating graded exercise test variables
affects the validity of the lactate threshold and
_VO2peak
Nicholas A. Jamnick1☯*, Javier Botella1☯, David B. Pyne2,3☯, David J. Bishop1,4☯
1 Institute for Health and Sport, College of Sport and Exercise Science, Victoria University, Melbourne,
Australia, 2 Australian Institute of Sport, Canberra, Australia, 3 Research Institute for Sport and Exercise
(UCRISE), University of Canberra, Canberra, Australia, 4 School of Medical and Health Sciences, Edith
Cowan University, Joondalup, Australia
☯ These authors contributed equally to this work.
* nicholas.jamnick@live.vu.edu.au
Abstract
Background
To determine the validity of the lactate threshold (LT) and maximal oxygen uptake ( _VO2max)
determined during graded exercise test (GXT) of different durations and using different LT
calculations. Trained male cyclists (n = 17) completed five GXTs of varying stage length (1,
3, 4, 7 and 10 min) to establish the LT, and a series of 30-min constant power bouts to estab-
lish the maximal lactate steady state (MLSS). _VO2 was assessed during each GXT and a
subsequent verification exhaustive bout (VEB), and 14 different LTs were calculated from
four of the GXTs (3, 4, 7 and 10 min)—yielding a total 56 LTs. Agreement was assessed
between the highest _VO2 measured during each GXT ( _VO2peak) as well as between each LT
and MLSS. _VO2peak and LT data were analysed using mean difference (MD) and intraclass
correlation (ICC).
Results
The _VO2peak value from GXT1 was 61.0 ± 5.3 mL.kg-1.min-1 and the peak power 420 ± 55 W
(mean ± SD). The power at the MLSS was 264 ± 39 W. _VO2peak from GXT3, 4, 7, 10 underesti-
mated _VO2peak by ~1–5 mL.kg-1.min-1. Many of the traditional LT methods were not valid and
a newly developed Modified Dmax method derived from GXT4 provided the most valid esti-
mate of the MLSS (MD = 1.1 W; ICC = 0.96).
Conclusion
The data highlight how GXT protocol design and data analysis influence the determination
of both _VO2peak and LT. It is also apparent that _VO2max and LT cannot be determined in a sin-
gle GXT, even with the inclusion of a VEB.
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OPEN ACCESS
Citation: Jamnick NA, Botella J, Pyne DB, Bishop
DJ (2018) Manipulating graded exercise test
variables affects the validity of the lactate threshold
and _VO2peak. PLoS ONE 13(7): e0199794. https://
doi.org/10.1371/journal.pone.0199794
Editor: Øyvind Sandbakk, Norwegian University of
Science and Technology, NORWAY
Received: April 30, 2018
Accepted: June 13, 2018
Published: July 30, 2018
Copyright: © 2018 Jamnick 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: The data underlying
this study have been uploaded to the Open Science
Framework and are accessible using the following
link: https://osf.io/293ns/.
Funding: Funding was provided by the Graduate
Research Office (PhD Student Budget) at Victoria
University. The funder had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
Introduction
Sampling of expired gas and blood data during a graded exercise test (GXT) to exhaustion per-
mits identification of the gas exchange threshold (GET), the respiratory compensation point
(RCP), the lactate threshold (LT), and maximal oxygen uptake ( _VO2max). These indices can dis-
tinguish cardiorespiratory fitness, and demarcate the domains of exercise [1, 2] that can be
used to prescribe exercise and to optimize training stimuli [3–6]. However, despite the popu-
larity of these indices, the methods used to determine them can differ substantially and there
has been little systematic investigation of their validity [7–9].
The recommended duration of a GXT to assess _VO2max is 8 to 12 minutes [10–13]. How-
ever, there is little consensus on an appropriate GXT protocol design, including duration,
stage length, or number of stages, needed to establish the LT. A stage length of at least 3 min-
utes has been recommended [13], although an 8-minute stage length has also been suggested
for blood lactate concentrations to stabilize [14]. The number of stages and GXT duration will
depend on the starting intensity and power increments. Power is typically increased identically
[15], regardless of sex or fitness, leading to a heterogenous GXT duration and number of stages
completed [16]. A customized approach to LT testing has been recommended to ensure a
more homogenous GXT duration [17].
More than 25 methods have been proposed to calculate the LT [18]; these include the
power preceding a rise in blood lactate concentration of more than 0.5, 1.0 or 1.5 mmol.L-1
from baseline [19], the onset of a fixed blood lactate accumulation (OBLA) ranging from
2.0 to 4.0 mmol.L-1 [20, 21], or the use of curve fitting procedures such as the Dmax or modi-
fied Dmax methods (ModDmax) [22, 23]. However, many of these ‘accepted’ methods are
influenced by GXT protocol design [8, 24] and their underlying validity has not been
reported.
Assessing the validity of a measurement requires comparison with a criterion measure.
The maximal lactate steady state (MLSS) represents the highest intensity where blood lac-
tate appearance and disappearance is in equilibrium and where energy demand is ade-
quately met by oxidative phosphorylation [25]. Exercise performed above the MLSS
results in accelerated blood lactate appearance and it has therefore been suggested as an
appropriate criterion measure for the LT [25, 26]. The primary advantages of the MLSS
test include its independence of participant effort, it’s submaximal and is reliable [27].
However, the disadvantage is the necessity of multiple laboratory visits and that it yields
only one index of performance.
_VO2max is considered the “gold standard” for assessing cardiorespiratory fitness [28]
and the highest recorded _VO2 from a GXT is often accepted as the _VO2max [10]. Establish-
ing the LT requires a GXT that typically exceeds 20 minutes [13]; however, in these
instances the highest _VO2 may underestimate the _VO2max [12] and is termed _VO2peak.
Recently, the use of a verification exhaustive bout (VEB) has been recommended to con-
firm the _VO2max. However, it is unknown if a VEB performed after a longer duration GXT
provides a valid estimate of _VO2max.
The aim of this study was to determine the validity of the LT and _VO2max derived from a sin-
gle visit GXT. We hypothesized that our results would yield one or more GXT stage length and
LT calculation method combination that provides a valid estimation of the criterion measure
of the LT (i.e., MLSS). We also hypothesized the highest _VO2 measured during longer duration
GXTs would underestimate _VO2max and that the highest _VO2 value measured during each VEB
would be similar to the _VO2peak measured during the 8- to 12-minute GXT.
Validation of a single visit graded exercise test
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Abbreviations: LT, lactate threshold; GXT, graded
exercise testing; _VO2max, maximal oxygen uptake;
OBLA, onset of fixed blood lactate accumulation;
MLSS, maximal lactate steady state; VEB,
verification exhaustive bout; BMI, body mass
index; _Wmax, maximum power output; _VO2pea,
highest measured oxygen uptake value; RCP,
respiratory compensation point; RCPMLSS, estimate
of the maximal lactate steady state via respiratory
compensation point; B + mmol.L-1, blood lactate
concentration increases above baseline value(s);
ModDmax, modified Dmax; Exp-Dmax, exponential
Dmax; Log-Poly-ModDmax, Log-log Modified Dmax;
Log-Exp-ModDmax, Log-log Exponential Modified
Dmax; CV, coefficient of the variation; ES, effect size;
r, Pearson product moment correlation; ICC,
intraclass correlation; SEM, standard error of the
measurement.
Materials and methods
Ethical approval
All procedures were performed in accordance with the ethical standards of the institutional
and/or national research committee, and with the 1964 Helsinki declaration and its later
amendments or comparable ethical standards.
Participants/Experimental design
Seventeen trained male cyclists ( _VO2max 62.1 ± 5.8 mL.kg-1.min-1, age 36.2 ± 7.4 years, body
mass index (BMI): 24.1 ± 2.0 kg.m-2) volunteered for this study which required 7 to 10 visits to
the laboratory. Informed consent was obtained from all individual participants included in the
study.
Visit one included risk stratification using the American College of Sports Medicine Risk
Stratification guidelines [29], written informed consent, self-reported physical activity rating
(PA-R) [30], measurement of height and body mass, and completion of a cycling GXT with
1-minute stages (GXT1) followed by a VEB. The remaining visits consisted of four cycling
GXTs with varying stage length (3-, 4-, 7- and 10-min stages) and a series of 30-min constant
power bouts to establish the MLSS. The GXTs and constant power bouts were performed in an
alternating order and the order of the GXTs was randomised. Prior to each GXT and the con-
stant power bouts a 5-min warm up was administered at a self-selected power followed by 5
min of passive rest. Participants performed each test at their preferred cadence determined
during the initial visit. Antecubital venous blood (1.0 mL) was sampled during all visits
(excluding GXT1) at rest, and at the end of every stage during the GXTs or every 5 min during
the constant power exercise bouts. All participants self-reported abstaining from the consump-
tion of alcohol and caffeine or engaging in heavy exercise 24 h prior to each visit. Participants
were given at least 48 h between visits and all tests were completed within 6 weeks. The Victo-
ria University Human Research Ethics Committee approved all procedures (HRE 017–035).
Equipment/Instruments
All exercise testing was conducted using an electronically-braked cycle ergometer (Lode Excal-
ibur v2.0, The Netherlands). A metabolic analyser (Quark Cardiopulmonary Exercise Testing,
Cosmed, Italy) was used to assess oxygen uptake ( _VO2) on a breath-by-breath basis, and heart
rate was measured throughout all tests. Antecubital venous blood was analysed using a blood
lactate analyser (YSI 2300 STAT Plus, YSI, USA).
GXTs with verification exhaustive bout
Demographic data, PA-R, and measurements of height and body mass were used to estimate
_VO2max [31] and maximum power output _Wmax [30, 32].
Est:VO2max ¼ 56:363 þ ð1:921 x PA were designed for each of the remaining GXTs based on a percentage of the measured _Wmax
from GXT1. The predicted _Wmax was 80%, 77%, 72% and 70% for GXT3, GXT4, GXT7, and
GXT10, respectively. The target number of stages for each participant was nine; the initial stage
and subsequent stages of the remaining GXTs were determined using the following equations:
Stage 1 Power ¼ Predicted _Wmax  0:25
Eq 3
Subsequent power increments ¼ ðPredicted _Wmax 6. Exponential Dmax (Exp-Dmax): The point on the exponential plus-constant regression curve
that yielded the maximum perpendicular distance to the straight line formed by the two
end points of the curve [41, 42].
7. Log-log Modified Dmax (Log-Poly-ModDmax): The intensity at the point on the third order
polynomial regression curve that yielded the maximal perpendicular distance to the straight
line formed by the intensity associated with the log-log LT and the final lactate point.
8. Log-log Exponential Modified Dmax method (Log-Exp-ModDmax): The intensity at the
point on the exponential plus-constant regression curve that yielded the maximal perpen-
dicular distance to the straight line formed by the intensity associated with the log-log LT
and the final lactate point.
9. RCP: refer to Constant Power Exercise Bouts to Establish the Maximal Lactate Steady State
method section.
10. The estimated MLSS was based on a regression equation based on the RCP from GXT1
(RCPMLSS) (Eq 5).
Data analysis
Breath-by-breath data were edited individually with values greater than three standard devia-
tions from the mean excluded [43]. The data was interpolated on a second-by-second basis
and averaged into 5- and 30-s bins [44, 45]. The highest measured _VO2 value from every GXT
and VEB was determined as the highest 20-s rolling average. The _VO2max was computed as the
Fig 1. Representative blood lactate curve with 14 LTs calculated from GXT4 (participant #9). The power of the MLSS was 302 W and the
blood lactate concentration was 2.85 mmol.L-1. Log-log = power at the intersection of two linear lines with the lowest residual sum of
squares; log = using the log-log method as the point of the initial data point when calculating the Dmax or Modified Dmax; poly = Modified
Dmax method calculated using a third order polynomial regression equation; exp = Modified Dmax method calculated using a constant plus
exponential regression equation; OBLA = onset of blood lactate accumulation; B + absolute value = the intensity where blood lactate
increases above baseline.
https://doi.org/10.1371/journal.pone.0199794.g001
Validation of a single visit graded exercise test
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highest _VO2 measured from any GXT or VEB. The _VO2peak for each GXT was defined as the
highest measured _VO2 from either the GXT or the subsequent VEB.
The _Wmax for every GXT was determined as the power from the last completed stage plus
the time completed in the subsequent stage multiplied by the slope (Eq 6). The _VO2 response
at the MLSS was determined by the average _VO2 value during the last two minutes of the
30-minute constant power bout.
_Wmax ¼ Power of Last Stage ðWÞ þ ½slope ðW:s Table 1. The mean ± standard deviation (SD) of the 14 lactate thresholds calculated from the 4 prolonged graded exercise tests (i.e., GXT3, GXT4, GXT7 and
GXT10), and the respiratory compensation point (RCP) and the maximal lactate steady state (MLSS) estimated from the RCP (RCPMLSS) calculated from GXT1.
GXT3
GXT4
GXT7
GXT10
Log-log LT
Mean SD (W)
211 ± 43
202 ± 38
200 ± 40
196 ± 41
MD (W)
53.1
62.8
64.8
68.3
r
0.84
0.89
0.87
0.78
ES
1.28
1.63
1.62
1.70
OBLA 2.0
Mean SD (W)
262 ± 40
249 ± 39
247 ± 39
245 ± 37
MD (W)
2.1
15.1
17.3
19.6
r
0.86
0.94
0.94
0.93
ES
-0.05
-0.38
-0.44
-0.50
OBLA 2.5
Mean SD (W)
276 ± 42
262 ± 40
258 ± 40
255 ± 38
MD (W)
-11.9
2.0
6.7
9.2
r
0.89
0.95
0.94
0.93
ES
0.30
-0.05
-0.17
-0.23
OBLA 3.0
Mean SD (W)
288 ± 43
273 ± 41
267 ± 41
264 ± 39
MD (W)
-23.2
-8.8
-2.2
0.4
r
0.90
0.96
0.95
0.93
ES
0.59
0.22
0.06
-0.01
OBLA 3.5
Mean SD (W)
297 ± 45
282 ± 41
274 ± 41
272 ± 40
MD (W)
-32.8
-18.1
-10.0
-7.3
r
0.91
0.96
0.95
0.93
ES
0.83
0.46
0.25
0.19
OBLA 4.0
Mean SD (W)
306 ± 46
291 ± 42
281 ± 42
279 ± 41
MD (W)
-41.3
-26.3
-16.8
-14.2
r
0.91
0.97
0.95
0.93
ES
1.05
0.67
0.43
0.36
Baseline + 0.5
Mean SD (W)
235 ± 38
229 ± 40
228 ± 41
225 ± 37
MD (W)
29.4
35.6
36.6
39.5
r
0.74
0.81
0.83
0.82
ES
-0.75
-0.90
-0.93
-1.00
Baseline + 1.0
Mean SD (W)
255 ± 39
239 ± 40
236 ± 39
235 ± 39
MD (W)
9.5
25.3
27.9
29.1
r
0.88
0.92
0.93
0.91
ES
-0.24
-0.64
-0.71
-0.74
Baseline + 1.5
Mean SD (W)
270 ± 41
254 ± 41
250 ± 39
248 ± 39
MD (W)
-6.0
10.1
14.7
16.8
r
0.90
0.94
0.94
0.92
ES
0.15
-0.26
-0.37
-0.43
Dmax
Mean SD (W)
246 ± 34
232 ± 36
223 ± 31
216 ± 33
MD (W)
18.6
31.9
41.6
48.8
r
0.94
0.97
0.96
0.95
ES
-0.47
-0.81
-1.06
-1.24
Modified Dmax
Mean SD (W)
278 ± 37
267 ± 39
255 ± 40
248 ± 37
MD (W)
-13.2
-2.9
9.7
15.9
r
0.90
0.91
0.93
0.92
ES
0.33
0.07
-0.25
-0.40
Log-Poly-MDmax
Mean SD (W)
280 ± 42
265 ± 42
255 ± 39
248 ± 40
MD (W)
-15.5
-1.1
9.5
16.5
(Continued)
Validation of a single visit graded exercise test
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total) and the MLSS (all log-log methods were excluded given an ES > 1.0). Ten of the calcu-
lated LTs and the RCPMLSS met our inclusion criteria for final analysis—detailed comparisons
with the MLSS are provided in Table 3 and Fig 3. Figs 3–7 shows Bland-Altman plots of the 11
estimations included in our analysis; the newly developed ModDmax LT calculations (Fig 5
Panel C and D; Fig 6 Panel C) had the lowest limits of agreement with the MLSS. The log-log
polynomial modified Dmax (Log-Poly-ModDmax) method derived from GXT4 provided the
best estimation of the MLSS (Fig 5 Panel C). There was an inverse relationship between the
power calculated for each of the 14 LTs and stage length (Tables 1 and 4).
_Wmax and _VO2max
There was an inverse relationship between GXT duration and both _Wmax and _VO2peak
(Table 5). The _VO2peak values derived from GXT3 and GXT4 were similar to the _VO2peak mea-
sured during GXT1 (Table 6); however, the values were outside the variability of the measure-
ment (CV > 3%) [27]. _VO2peak values from GXT1 and the corresponding VEB had the highest
agreement (MD = 0.5 mL.kg-1.min-1, ICC = 0.96, SEM = 1.1 mL.kg-1.min-1 and CV = 2.0%)
compared with any GXT and corresponding VEB. The remaining GXTs and corresponding
Table 1. (Continued)
GXT3
GXT4
GXT7
GXT10
r
0.94
0.96
0.96
0.92
ES
0.39
0.03
-0.24
-0.42
Exp-Dmax
Mean SD (W)
256 ± 35
243 ± 36
234 ± 34
228 ± 35
MD (W)
8.0
21.8
30.8
36.8
r
0.92
0.97
0.96
0.94
ES
-0.20
-0.55
-0.78
-0.93
Log-Exp-MDmax
Mean SD (W)
286 ± 42
271 ± 42
260 ± 39
253 ± 40
MD (W)
-21.7
-7.0
4.3
11.1
r
0.94
0.97
0.96
0.93
ES
0.55
0.18
-0.11
-0.28
GXT1
RCPMLSS
Mean SD (W)
271 ± 39
MD (W)
-6.71
r
0.92
ES
-0.17
RCP
Mean SD (W)
315 ± 40
MD (W)
-50.4
r
0.91
ES
1.27
https://doi.org/10.1371/journal.pone.0199794.t001
Table 2. Mean, standard deviation, and range of the _VO2 and power associated with the maximal lactate steady state (MLSS) expressed as a percentage of the maxi-
mal power ( _Wmax) and _VO2peak measured during each GXT. Note: The _VO2 at the MLSS was 81.4 ± 4.7% of the _VO2max. (Defined as the highest measured _VO2 during
any GXT).
GXT1
GXT3
GXT4
GXT7
GXT10
_VO2 at MLSS
(% of _VO2peak)
83.0 ± 4.5
[75.5–90.7]
84.7 ± 4.7
[76.6–91.9]
86.1 ± 5.9
[73.9–94.2]
88.4 ± 6.0
[77.4–103.2]
90.2 ± 5.3
[78.7–99.9]
Power at MLSS
(% of _Wmax)
62.9 ± 3.9
[56.8–71.7]
78.4 ± 4.3
[69.8–84.4]
82.4 ± 3.6
[73.7–88.8]
87.3 ± 4.4
[79.8–96.0]
89.6 ± 4.7
[81.6–98.1]
https://doi.org/10.1371/journal.pone.0199794.t002
Validation of a single visit graded exercise test
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Validation of a single visit graded exercise test
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July 30, 2018
9 / 21
VEB had a CV of 3.3, 2.0, 3.5 and 5.2%, for GXT3, GXT4, GXT7 and GXT10, respectively. The
VEB performed following the longer duration GXTs (GXT3-10) underestimated the _VO2peak
from GXT1 (Table 6).
Discussion
The main findings of the present study are as follows. Only 11 of the 58 threshold values met
our inclusion criteria as valid estimates of the MLSS. Of the 11 methods included in our analy-
sis, three of the ModDmax methods yielded the most favourable estimations of the MLSS, and
the Log-Poly-ModDmax derived from GXT4 provided the best estimation of the MLSS. There
was an inverse relationship between stage length and LT, and this effect was larger in all Dmax
methods compared with the OBLA and baseline plus absolute lactate value methods. The
_VO2peak values measured during the longer duration GXTs (GXT3-10) underestimated the
_VO2max and the _VO2peak values obtained from GXT1 (MD = 1.2 to 4.8 mL.kg-1.min-1). Finally,
contrary to our hypothesis, the VEB after the longer duration GXTs did not yield _VO2peak val-
ues comparable to the _VO2peak derived from GXT1.
The use of five GXT protocols, 14 common LT methods, the RCP and RCPMLSS resulted in
58 unique thresholds. However, despite their common use, we observed that only 11 of these
values met our criteria for inclusion (MD < 7.9 W; ES < 0.2; r > 0.90). Of the four Dmax meth-
ods included in our analysis, one consisted of the traditional ModDmax method [22]. This had
the poorest agreement relative to the other ModDmax methods included in our analysis. The
remaining three Dmax methods are new variations of the ModDmax method, and the Log-Poly-
Fig 2. (A-D) Forrest Plots of the difference (ES ± 95% CI) between the MLSS and the power calculated from the 13
lactate thresholds derived from (A) GXT3, (B) GXT4, (C) GXT7 and (D) GXT10 (52 in total and excluding log-log). The
solid vertical bar represents no difference from the MLSS and the dashed vertical bars represents the threshold between
a trivial and small difference (ES = 0.2) established by Cohen (50) and Hopkins (49). log = using the log-log method as
the initial data point when calculating the Dmax or Modified Dmax; poly = Modified Dmax method calculated using a
third order polynomial regression equation; exp = Modified Dmax method calculated using a constant plus exponential
regression equation; OBLA = onset of blood lactate accumulation.
https://doi.org/10.1371/journal.pone.0199794.g002
Table 3. Mean ± standard deviation, mean difference (MD), intraclass correlation coefficient (ICC), Lin’s concordance correlation coefficient (ρc), standard error
of the measurement (SEM), effect size (ES) with 95% confidence limits, and coefficient of the variation (%CV) between the maximal lactate steady state (MLSS) and
the eleven thresholds included in our analysis. (RCPMLSS = MLSS estimate based on the respiratory compensation point; log = Modified Dmax method using the log-log
method as the point of the initial lactate point; poly = Modified Dmax method calculated using a third order polynomial regression equation; exp = Modified Dmax method
calculated using a constant plus exponential regression equation; OBLA = onset of blood lactate accumulation).
Mean ± SD
(W)
MD
(W)
ICC [95% CI]
ρc
SEM [95% CI]
(W)
ES [95% CI]
CV [95% CI] (%)
MLSS
264 ± 39
GXT1
RCPMLSS
271 ± 39
6.7
0.92 [0.78–0.97]
0.90
11.2 [8.3–17.0]
0.17 [-0.04–0.38]
6.0 [4.4–9.4]
GXT3
Baseline + 1.5 mmol.L-1
270 ± 41
6.0
0.90 [0.75–0.97]
0.90
12.5 [9.3–19.0]
0.15 [-0.08–0.38]
6.6 [4.9–10.4]
GXT4
OBLA 2.5 mmol.L-1
262 ± 40
-2.0
0.95 [0.87–0.98]
0.95
8.7 [6.5–13.2]
-0.05 [-0.21–0.11]
5.3 [3.9–8.4]
Modified Dmax
267 ± 39
2.9
0.91 [0.76–0.98]
0.90
11.7 [8.7–17.9]
0.07 [-0.15–0.29]
7.0 [5.1–11.0]
Log-Poly-MDmax
265 ± 42
1.1
0.96 [0.90–0.99]
0.96
7.9 [5.8–12.0]
0.03 [-0.11–0.17]
4.4 [3.2–6.9]
Log-Exp-MDmax
271 ± 42
7.0
0.97 [0.91–0.99]
0.95
7.5 [5.6–11.4]
0.18 [0.04–0.32]
4.1 [3.0–6.3]
GXT7
OBLA 2.5 mmol.L-1
258 ± 41
-6.7
0.94 [0.85–0.98]
0.93
9.4 [7.0–14.3]
-0.17 [-0.34–0.00]
4.9 [3.6–7.7]
OBLA 3.0 mmol.L-1
267 ± 41
2.2
0.95 [0.86–0.98[
0.95
9.2 [6.9–14.1]
0.06 [-0.11–0.23]
5.1 [3.7–8.0]
Log-Exp-MDmax
260 ± 39
-4.3
0.96 [0.89–0.99]
0.95
7.8 [5.8–11.9]
-0.11 [-0.25–0.03]
4.1 [3.0–6.4]
GXT10
OBLA 3.0 mmol.L-1
264 ± 39
-0.4
0.93 [0.82–0.98]
0.93
10.2 [7.6–15.5]
-0.01 [-0.20–0.18]
5.5 [4.0–8.6]
OBLA 3.5 mmol.L-1 (n = 16)
275 ± 39
6.9
0.93 [0.82–0.98]
0.91
10.3 [7.7–15.7]
0.19 [0.00–0.38]
5.5 [4.0–8.7]
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ModDmax derived from GXT4 had the highest correlation and lowest mean difference with the
MLSS. These variations of the ModDmax method use the power at the log-log LT as the initial
intensity to calculate the ModDmax and then either the traditional third-order polynomial or
exponential plus-constant regression curve to fit the lactate curve [23, 41]. Although the valid-
ity of these three methods has not previously been assessed, the favourable estimations of the
MLSS may be related to the greater objectivity with which they determine the intensity that
corresponds with the initial rise in blood lactate concentration [37].
Fig 3. Bland-Altman plots displaying agreement between measures of the power associated with the RCP
regression equation (RCPMLSS) calculated from GXT1 and the MLSS. The differences between measures (y-axis) are
plotted as a function of the mean of the two measures (x-axis) in power (Watts). The horizontal solid line represents
the mean difference between the two measures (i.e., bias). The two horizontal dashed lines represent the limits of
agreement (1.96 x standard deviation of the mean difference between the estimated lactate threshold via the RCPMLSS
and the maximal lactate steady state). The dotted diagonal lines represent the boundaries of the 95% CI for MLSS
reliability (CV = 3.0%; 95%; CI = 3.8%) calculated from Hauser et al., 2014) (RCP = respiratory compensation point).
https://doi.org/10.1371/journal.pone.0199794.g003
Fig 4. Bland-Altman plots displaying agreement between measures of the power associated with the baseline plus
1.5 mmol.L-1 calculated from GXT3 and the MLSS. The differences between measures (y-axis) are plotted as a
function of the mean of the two measures (x-axis) in power (Watts). The horizontal solid line represents the mean
difference between the two measures (i.e., bias). The two horizontal dashed lines represent the limits of agreement
(1.96 x standard deviation of the mean difference between the lactate threshold and the maximal lactate steady state).
The dotted diagonal lines represent the boundaries of the 95% CI for MLSS reliability (CV = 3.0%; 95%; CI = 3.8%)
calculated from Hauser et al., 2014).
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Although the original Dmax method is a commonly cited method for determining the LT
[23], we observed large mean differences (19 to 49 W) between the Dmax and MLSS. Three pre-
vious studies have purported to investigate the validity of this method to estimate the MLSS in
trained male cyclists [15, 52, 53]. One concluded that the Dmax method derived from GXT3
was a valid estimation of the MLSS (r = 0.97) [54]. We also observed a high correlation
between Dmax and the MLSS (r = 0.94 to 0.97) (Table 1), but, as indicated by the MD and other
measures, a high correlation is not sufficient to establish validity [55]. Another study examined
Dmax derived from two GXTs with similar durations (36 vs. 39 min), but with different stage
lengths (30-s vs. 6-min) [15]. The Dmax derived from GXT30s was not correlated (r = 0.51) with
the MLSS, even though the MD was 5 W, whilst the Dmax derived from GXT6 was correlated
(r = 0.85); however, it underestimated the MLSS (MD = 22 W). The third study concluded the
Dmax derived from GXT1 yielded poor estimates of the MLSS (r = 0.56; bias = -1.8 ± 38.1 W)
[53]. Thus, although some studies [15, 54] have used correlation analysis to suggest the Dmax
provides a valid estimate of the MLSS, this is not supported by the more comprehensive assess-
ment of validity performed in the present and other studies [53].
There were five fixed blood LT methods and one baseline plus an absolute value that met
our inclusion criteria, and, as previously reported [15, 24], these varied with the GXT protocol
used. The baseline + 1.5 mmol.L-1 was the only LT derived from GXT3 included in our analysis
(bias = -6 ± 35 W). This is consistent with the results of one previous study (bias = 0.5 ± 24
W), which also recruited trained male cyclists and had a similar GXT protocol design [56].
Consistent with our findings, this study also reported that an OBLA of 3.5 mmol.L-1 derived
from GXT3 did not provide a valid estimation of the MLSS. In contrast, another study con-
firmed the validity of the OBLA of 3.5 mmol.L-1 [52], despite recruiting trained cyclists and
using an identical GXT protocol. These conflicting results are likely attributable to the low
reproducibility of the OBLA methods [16].
While none of the OBLAs from GXT3 met our inclusion criteria, the OBLA methods of 2.5
mmol.L-1 derived from GXT4 and GXT7 provided valid estimations of the MLSS, as did the
OBLA of 3.0 mmol.L-1 derived from GXT7 and GXT10. The OBLA of 3.5 mmol.L-1 from
GXT10 was the highest fixed blood LT that identified the MLSS. There is no previous data
investigating the validity of these OBLA methods. However, it is worth noting that these five
methods provided superior estimations of the MLSS compared with the original ModDmax,
but were less favourable than the newly-developed ModDmax methods.
An OBLA of 4.0 mmol.L-1 is the most commonly-accepted fixed blood lactate value for esti-
mating the LT or MLSS. Three previous studies have attempted to validate use of an OBLA of
4.0 mmol.L-1 with cycle ergometry [15, 53, 57]. One study found that it overestimated the
MLSS (MD = 49 W) when derived from GXT1 [53]. The other study reported poor agreement
(bias 7 ± 49 W) when OBLA of 4.0 mmol.L-1 was derived from GXT4 [57]. The final study
observed a poor correlation between an OBLA of 4.0 mmol.L-1 and the MLSS (r = 0.71) [15].
Our results indicated the OBLA of 4.0 mmol.L-1 overestimated the MLSS across all GXTs.
Fig 5. (A-D) Bland-Altman plots displaying agreement between measures of the power associated with the (A) OBLA
2.5 mmol.L-1, (B) Modified Dmax, (C) Log-Poly-Modified Dmax, (D) Log-Exp-Modified Dmax calculated from GXT4
and the MLSS. The differences between measures (y-axis) are plotted as a function of the mean of the two measures (x-
axis) in power (Watts). The horizontal solid line represents the mean difference between the two measures (i.e., bias).
The two horizontal dashed lines represent the limits of agreement (1.96 x standard deviation of the mean difference
between the lactate threshold and the maximal lactate steady state). The dotted diagonal lines represent the boundaries
of the 95% CI for MLSS reliability (CV = 3.0%; 95%; CI = 3.8%) calculated from Hauser et al., 2014) (log = Modified
Dmax method using the log-log method as the point of the initial lactate point; poly = Modified Dmax method calculated
using a third order polynomial regression equation; exp = Modified Dmax method calculated using a constant plus
exponential regression equation; OBLA = onset of blood lactate accumulation.).
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Fig 6. (A-C) Bland-Altman plots displaying agreement between measures of the power associated with the (A) OBLA
2.5 mmol.L-1 (GXT7), (B) OBLA 3.0 mmol.L-1 (GXT7), (C) Log-Exp-Modified Dmax calculated from GXT7 and the
MLSS. The differences between measures (y-axis) are plotted as a function of the mean of the two measures (x-axis) in
power (Watts). The horizontal solid line represents the mean difference between the two measures (i.e., bias). The two
horizontal dashed lines represent the limits of agreement (1.96 x standard deviation of the mean difference between the
lactate threshold and the maximal lactate steady state). The dotted diagonal lines represent the boundaries of the 95%
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Thus, in agreement with previous research, our results indicate; the OBLA of 4.0 mmol.L-1
does not accurately estimate the MLSS. It is also worth noting that the original authors cau-
tioned the use of this OBLA method, given the lack of a significant correlation when compar-
ing OBLA methods from a GXT and the MLSS [24].
The RCP derived from an 8- to 12-minute GXT consistently overestimates the MLSS [44,
53], and this was confirmed in our study (Table 1). Therefore, we used a regression equation
based on the RCP (RCPMLSS) (Eq 5) to estimate the starting intensity for establishing the
MLSS [33]. Our results indicate there was good agreement between the MLSS and RCPMLSS
CI for MLSS reliability (CV = 3.0%; 95%; CI = 3.8%) calculated from Hauser et al., 2014) (log = Modified Dmax method
using the log-log method as the point of the initial lactate point; exp = Modified Dmax method calculated using a
constant plus exponential regression equation; OBLA = onset of blood lactate accumulation.).
https://doi.org/10.1371/journal.pone.0199794.g006
Fig 7. (A-B) Bland-Altman plots displaying agreement between measures of the power associated with the (A) OBLA
3.0 mmol.L-1, (B) OBLA 3.5 mmol.L-1 calculated from GXT10 and the MLSS. The differences between measures (y-
axis) are plotted as a function of the mean of the two measures (x-axis) in power (Watts). The horizontal solid line
represents the mean difference between the two measures (i.e., bias). The two horizontal dashed lines represent the
limits of agreement (1.96 x standard deviation of the mean difference between the lactate threshold and the maximal
lactate steady state). The dotted diagonal lines represent the boundaries of the 95% CI for MLSS reliability (CV = 3.0%;
95%; CI = 3.8%) calculated from Hauser et al., 2014) (OBLA = onset of blood lactate accumulation.).
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(Table 3). Nonetheless, for many participants the difference between MLSS and RCPMLSS
exceeded the CV% for the MLSS (Fig 3). Therefore, although the RCPMLSS can be used as a
convenient ‘starting point’ when establishing the MLSS, we recommend methods based on
blood sampling from the current study and assessing blood lactate kinetics in real time as rec-
ommended by Hering et al. [58] for a more accurate estimation of the MLSS.
Table 4. Mean difference (MD), effect size (ES), and p-value comparing the influence of graded exercise test stage length on all 14 lactate threshold methods.
3 vs. 4
3 vs. 7
3 vs. 10
4 vs. 7
4 vs. 10
7 vs. 10
Log-log LT
MD (W)
10
12
15
2
6
3
ES
0.24
0.28
0.36
0.05
0.14
0.08
p-value
0.09
0.02
0.02
0.63
0.15
0.47
OBLA 4.0 mmol.L-1
MD (W)
15
24
27
9
12
3
ES
0.34
0.56
0.63
0.22
0.29
0.06
p-value
0.00
0.00
0.00
0.05
0.01
0.35
OBLA 3.5 mmol.L-1
MD (W)
15
23
25
8
11
3
ES
0.34
0.53
0.60
0.20
0.26
0.06
p-value
0.00
0.00
0.00
0.09
0.02
0.35
OBLA 3.0 mmol.L-1
MD (W)
14
21
24
7
9
3
ES
0.34
0.50
0.57
0.16
0.23
0.06
p-value
0.00
0.00
0.00
0.16
0.05
0.36
OBLA 2.5 mmol.L-1
MD (W)
14
19
21
5
7
2
ES
0.34
0.46
0.53
0.12
0.18
0.06
p-value
0.00
0.00
0.00
0.30
0.13
0.39
OBLA 2.0 mmol.L-1
MD (W)
13
15
18
2
4
2
ES
0.33
0.38
0.45
0.06
0.12
0.06
p-value
0.01
0.01
0.00
0.63
0.36
0.45
Baseline + 0.5 mmol.L-1
MD (W)
6
7
10
1
4
3
ES
0.16
0.18
0.27
0.03
0.10
0.07
p-value
0.25
0.27
0.10
0.85
0.46
0.50
Baseline + 1.0 mmol.L-1
MD (W)
16
18
20
3
4
1
ES
0.40
0.47
0.51
0.07
0.10
0.03
p-value
0.01
0.00
0.00
0.53
0.41
0.71
Baseline + 1.5 mmol.L-1
MD (W)
16
21
23
5
7
2
ES
0.39
0.52
0.57
0.12
0.17
0.05
p-value
0.00
0.00
0.00
0.27
0.14
0.49
Dmax
MD (W)
13
23
30
10
17
7
ES
0.38
0.71
0.90
0.29
0.49
0.22
p-value
0.00
0.00
0.00
0.00
0.00
0.00
Modified Dmax
MD (W)
10
23
29
13
19
6
ES
0.27
0.59
0.79
0.32
0.50
0.16
p-value
0.01
0.00
0.00
0.01
0.00
0.06
Log-Poly-ModDmax
MD (W)
14
25
32
11
18
7
ES
0.35
0.62
0.78
0.26
0.43
0.18
p-value
0.00
0.00
0.00
0.00
0.00
0.02
Exp-Dmax
MD (W)
14
23
29
9
15
6
ES
0.38
0.66
0.82
0.26
0.42
0.17
p-value
0.00
0.00
0.00
0.00
0.00
0.02
Log-Exp-ModDmax
MD (W)
15
26
33
11
18
7
ES
0.35
0.64
0.80
0.28
0.44
0.17
p-value
0.00
0.00
0.00
0.00
0.00
0.01
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Although a single GXT can be used to estimate both _VO2max and LT, the optimal test dura-
tion for each measure is different [11, 13]. To address this challenge, we added a supramaximal
VEB after each GXT, equivalent to that performed following GXT1, expecting all VEBs would
yield similar _VO2 values. However, the _VO2peak values from the VEB after the longer duration
GXTs underestimated the _VO2peak from GXT1. Although the _VO2peak values from GXT3 and
GXT4 were similar to GXT1, the differences were larger than the typical coefficient of variabil-
ity for _VO2peak (CV < 3%) [59]. Our results are consistent with previous recommendations
that longer duration GXTs are not optimal for establishing _VO2peak [10, 60]. Furthermore,
while a VEB can be used to verify that _VO2peak was achieved, it appears that a VEB following a
prolonged GXT cannot be used to establish _VO2max.
Extending the duration of the GXT stages results in a lower _Wmax [61]. This has implica-
tions for exercise prescription, as it is common in sport and exercise science research to pre-
scribe exercise intensity as a percentage of _Wmax. For example, in the present study the MLSS
ranged from 63 ± 4% (range = 52 to 72%) of _Wmax from GXT1 to 82 ± 4% (range = 74 to 88%)
Table 5. Mean and standard deviation of _VO2max—highest measured _VO2 during any graded exercise test (GXT); GXT _VO2 -highest measured _VO2 during each
GXT; VEB _VO2 highest measured _VO2 during each verification exhaustive bout (VEB); _VO2peak, highest measured _VO2 during either the GXT or corresponding
VEB. Mean and standard deviation of GXT duration, max power (Watts) from each GXT, percentage of maximum power from the prolonged GXT expressed as a per-
centage of W maximum power from GXT1 and power of each VEB (Watts) from the GXTs. Relative power of the verification exhaustive bout expressed as a percentage
(%) of the maximal power measured during the GXT. The subscript (i.e., 1, 3, 4, 7 or 10) refers to the stage duration (minutes) for each test.
GXT1
GXT3
GXT4
GXT7
GXT10
_VO2max (mL.kg-1.min-1)
62.1 ± 5.8
GXT _VO2 (mL.kg-1.min-1)
60.6 ± 5.4
58.2 ± 5.3
57.3 ± 5.7
56.4 ± 5.2
54.9 ± 4.9
VEB _VO2 (mL.kg-1.min-1)
60.1 ± 5.8
58.9 ± 5.9
58.8 ± 6.1
56.4 ± 5.9
54.7 ± 6.6
_VO2peak (mL.kg-1.min-1)
61.0 ± 5.3
59.7 ± 5.4
58.9 ± 6.0
57.3 ± 5.4
56.2 ± 5.5
GXT Duration (min)
11.3 ± 0.9
26.8 ± 1.4
34.9 ± 1.9
59.2 ± 3.3
81.6 ± 4.6
Maximum Power (Watts)
420 ± 55
337 ± 46
321 ± 47
303 ± 43
295 ± 43
Percent _Wmax of GXT1 (%)
100
80.3 ± 2.9
76.4 ± 3.1
72.1 ± 3.6
70.3 ± 4.0
VEB (Watts)
378 ± 50
VEB (% of GXT _Wmax)
90
109.7 ± 3.8
118.4 ± 18.7
125.4 ± 19.3
128.8 ± 20.4
https://doi.org/10.1371/journal.pone.0199794.t005
Table 6. Mean difference (MD) and standard deviation, effect size (ES), coefficient of the variation (CV) and p-value (p) for the measured _VO2peak values from
GXT1 compared with the _VO2peak values from GXT3, GXT4, GXT7, and GXT10 and for the _VO2peak values from GXT1 compared with the _VO2peak values from the
VEB following GXT3, GXT4, GXT7, and GXT10. The subscript (i.e., 1, 3, 4, 7 or 10) refers to the stage duration (minutes) for each test.
GXT1 vs. GXT3
GXT1 vs. GXT4
GXT1 vs. GXT7
GXT1 vs. GXT10
MD (mL.kg-1.min-1)
-1.2 ± 3.3
-2.1 ± 4.2
-3.7 ± 4.7
-4.8 ± 3.7
ES
0.23
0.36
0.69
0.88
CV (%)
3.8
4.9
5.6
4.6
p
0.13
0.06
< 0.01
< 0.01
GXT1 vs. VEB GXT3
GXT1 vs. VEB GXT4
GXT1 vs. VEB GXT7
GXT1 vs. VEB GXT10
MD (mL.kg-1.min-1)
-2.1 ± 5.9
-2.1 ± 6.1
-4.6 ± 5.9
-6.2 ± 6.6
ES
0.37
0.37
0.81
1.04
CV (%)
4.2
4.9
6.1
5.9
p
0.02
0.98
0.03
0.03
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of _Wmax from GXT4. Prescribing exercise in the current study cohort at a fixed percentage of
_Wmax (e.g., 73% of _Wmax), would result in all participants exercising above or below the MLSS,
GXT1 and GXT4, respectively. This is important as it has previously been reported that pre-
scribing exercise relative to LT results in a more homogenous physiological response than
when exercise performed relative to _Wmax [62]. This also highlights why it is important to con-
sider the GXT protocol and the method used to determine relative exercise intensity when
comparing results between studies.
The wide range of _Wmax for each GXT is also note-worthy, the _Wmax range for GXT1 was
320 to 517 W and the duration ranged from 9 to 12 minutes. Had we employed a standardized
GXT (e.g., 35 W increments), and assuming _Wmax stayed constant, the range would have been
9- to 15 min. Applying this to our longer duration GXTs resulted in a homogenous duration
(GXT4: 32- to 39 min), whereas a standardised approach (e.g., 35 W increments) would have
resulted in a range of 27- to 46 min [57]. Thus, individualizing GXT protocol design is a useful
approach to ensure homogenous test duration [17].
Conclusion
In conclusion, the traditional Dmax and OBLA of 4.0 mmol.L-1 did not provide valid estimates
of the MLSS. The best estimation of the MLSS was the Log-Poly-ModDmax derived from
GXT4. The validity of our newly-developed ModDmax model may relate to the objectivity for
determining the initial rise in blood lactate concentration. However, we must advise caution
with the use of our newly-developed method until future research investigates the reliability
and reproducibility. It is apparent that both _VO2max and LT cannot be determined in a single
GXT, even if the GXT is followed by a VEB. Therefore, to appropriately determine _VO2max the
optimum duration of a GXT is 8–12 minutes and the _VO2 values measured during the GXT
and VEB be within 3% = CV [63]. Our data also highlight how differences in GXT protocol
design and methods used to calculate the relative exercise intensity may contribute to the con-
flicting findings reported in the literature.
Author Contributions
Conceptualization: Nicholas A. Jamnick, Javier Botella, David B. Pyne, David J. Bishop.
Data curation: Nicholas A. Jamnick, Javier Botella, David B. Pyne, David J. Bishop.
Formal analysis: Nicholas A. Jamnick, Javier Botella, David B. Pyne, David J. Bishop.
Investigation: Nicholas A. Jamnick, Javier Botella, David J. Bishop.
Methodology: Nicholas A. Jamnick, Javier Botella, David B. Pyne, David J. Bishop.
Project administration: Nicholas A. Jamnick, David J. Bishop.
Resources: Nicholas A. Jamnick, Javier Botella, David J. Bishop.
Software: Nicholas A. Jamnick.
Supervision: David B. Pyne.
Validation: Nicholas A. Jamnick, Javier Botella, David J. Bishop.
Visualization: Javier Botella.
Writing – original draft: Nicholas A. Jamnick, Javier Botella, David B. Pyne, David J. Bishop.
Validation of a single visit graded exercise test
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Writing – review & editing: Nicholas A. Jamnick, Javier Botella, David B. Pyne, David J.
Bishop.
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Manipulating graded exercise test variables affects the validity of the lactate threshold and [Formula: see text].
Jamnick, Nicholas A,Botella, Javier,Pyne, David B,Bishop, David J