Sao Paulo Med J. 2016; 134(3):193-8     193
ORIGINAL ARTICLE
DOI: 10.1590/1516-3180.2014.8921512
Post-analysis methods for lactate threshold depend on 
training intensity and aerobic capacity in runners.  
An experimental laboratory study
Métodos de pós-análise do limiar do lactato dependem da intensidade de treinamento 
e da capacidade aeróbica dos corredores. Um estudo laboratorial experimental
Tiago Lazzaretti FernandesI, Rômulo dos Santos Sobreira NunesII, Cesar Cavinato Cal AbadIII, Andrea Clemente Baptista SilvaIV, 
Larissa Silva SouzaIV, Paulo Roberto Santos SilvaV, Cyro AlbuquerqueVI, Maria Cláudia IrigoyenVII, Arnaldo José HernandezVIII
Laboratório do Estudo do Movimento (LEM), Instituto de Ortopedia e Traumatologia (IOT), Hospital das Clínicas (HC), Faculdade de Medicina da 
Universidade de São Paulo (FMUSP), and Sports Medicine Group of FMUSP, FIFA Medical Centre of Excellence, IOT HC-FMSUP, São Paulo, Brazil
ABSTRACT
CONTEXT AND OBJECTIVE: This study aimed to evaluate different mathematical post-analysis methods 
of determining lactate threshold in highly and lowly trained endurance runners.
DESIGN AND SETTING: Experimental laboratory study, in a tertiary-level public university hospital.
METHOD: Twenty-seven male endurance runners were divided into two training load groups: lowly 
trained (frequency < 4 times per week, < 6 consecutive months, training velocity ≥ 5.0 min/km) and 
highly trained (frequency ≥ 4 times per week, ≥ 6 consecutive months, training velocity < 5.0 min/km). 
The subjects performed an incremental treadmill protocol, with 1 km/h increases at each subsequent 
4-minute stage.  Fingerprint  blood-lactate analysis was performed at the end of each stage. The lactate 
threshold (i.e. the running velocity at which blood lactate levels began to exponentially increase) was 
measured using three different methods: increase in blood lactate of 1 mmol/l at stages (DT1), absolute 4 
mmol/l blood lactate concentration (4 mmol), and the semi-log method (semi-log). ANOVA was used to 
compare different lactate threshold methods and training groups. 
RESULTS: Highly trained athletes showed significantly greater lactate thresholds than lowly trained run-
ners, regardless of the calculation method used. When all the subject data were combined, DT1 and semi-
log were not different, while 4 mmol was significantly lower than the other two methods. These same 
trends were observed when comparing lactate threshold methods in the lowly trained group. However, 4 
mmol was only significantly lower than DT1 in the highly trained group. 
CONCLUSION: The 4 mmol protocol did not show  lactate threshold measurements comparable with DT1 
and semi-log protocols among lowly trained athletes.
RESUMO
CONTEXTO E OBJETIVO: O objetivo do presente estudo é avaliar modelos matemáticos de pós-análise 
do limiar de lactato em grupos de corredores de longa distância muito ou pouco treinados. 
TIPO DE ESTUDO E LOCAL: Estudo laboratorial experimental. Hospital Público Universitário Terciário.
MÉTODO: Vinte e sete corredores homens foram divididos em: pouco treinados (frequência < 4 vezes 
por semana, < 6 meses, velocidade ≥ 5,0 minutos/km) e muito treinados (frequência ≥ 4 vezes por sema-
na, ≥ 6  meses, velocidade < 5,0 minutos/km). Os participantes foram submetidos a protocolo de esteira 
escalonado (1% inclinação) = 1 km/h por fase (4 minutos). Ao fim de cada estágio, análise da “impressão 
digital” metabolômica foi realizada. O limiar do lactato (i.e. velocidade em que o lactato sanguíneo aumen-
ta exponencialmente) foi medido utilizando-se três métodos: aumento de 1 mmol/l da concentração, 
concentração absoluta de 4 mmol e método semi-log. ANOVA foi utilizada para comparar os diferentes 
limiares de lactato e grupos.
RESULTADO: Atletas muito treinados apresentaram limiares de lactato maiores que os corredores pouco 
treinados, independentemente do método de cálculo utilizado. Comparando todos os corredores juntos, 
as análises de aumento de 1 mmol/l e semi-log não foram diferentes, enquanto a concentração absoluta 
de 4 mmol/l foi significativamente mais baixa que as dos dois outros métodos. Essas mesmas tendências 
foram observadas ao se compararem os métodos de limiar de lactato no grupo menos treinado. Entretan-
to, a análise absoluta de 4 mmol/l foi menor do que a do aumento de 1 mmol/l no grupo muito treinado. 
CONCLUSÃO: O método concentração absoluta de 4 mmol não mostrou mensurações comparáveis de 
limiar do lactato quando comparado com os protocolos aumento de 1 mmol/l e semi-log nos atletas 
pouco treinados. 
IMD, MSc. Doctoral Student and Attending 
Physician, Sports Medicine Group, FIFA Medical 
Centre of Excellence, Faculdade de Medicina 
da Universidade de São Paulo (FMUSP), and 
Instituto de Ortopedia e Traumatologia (IOT), 
Hospital das Clínicas (HC), São Paulo, Brazil.
IIUndergraduate Student, Faculdade de Medicina da 
Universidade de São Paulo (FMUSP), São Paulo, Brazil.
IIIMSc, PhD. Heart Institute, Faculdade de Medicina 
da Universidade de São Paulo (FMUSP), and 
Instituto do Coração (InCor), São Paulo, Brazil.
IVMD. Sports Medicine Group, Faculdade de 
Medicina da Universidade de São Paulo (FMUSP), 
and Instituto de Ortopedia e Traumatologia (IOT), 
Hospital das Clínicas (HC), São Paulo, Brazil.
VPhD. Sports Medicine Group, FIFA Medical 
Centre of Excellence, Faculdade de Medicina 
da Universidade de São Paulo (FMUSP), and 
Instituto de Ortopedia e Traumatologia (IOT), 
Hospital das Clínicas (HC), São Paulo, Brazil.
VIMSC, PhD. Assistant Professor, Department of 
Mechanical Engineering, Centro Universitário da 
FEI, São Bernando do Campo, Brazil.
VIIMD, PhD. Professor, Faculdade de Medicina 
da Universidade de São Paulo (FMUSP), and 
Instituto do Coração (InCor), São Paulo, Brazil.
VIIIPhD. Assistant Professor, Director of Sports 
Medicine Group, FIFA Medical Centre of 
Excellence, Faculdade de Medicina da 
Universidade de São Paulo (FMUSP), and 
Instituto de Ortopedia e Traumatologia (IOT), 
Hospital das Clínicas (HC), São Paulo, Brazil.
KEY WORDS:
Lactic acid.
Physical endurance.
Anaerobic threshold.
Oxygen consumption.
Exercise test.
Sports medicine.
PALAVRAS-CHAVE:
Ácido láctico.
Resistência física.
Limiar anaeróbio.
Consumo de oxigênio.
Teste de esforço.
Medicina esportiva. 
ORIGINAL ARTICLE | Fernandes TL, Nunes RSS, Abad CCC, Silva ACB, Souza LS, Silva PRS, Albuquerque C, Irigoyen MC, Hernandez AJ
194     Sao Paulo Med J. 2016; 134(3):193-8
INTRODUCTION
Blood lactate evaluation commonly complements endurance 
training regimens.1,2 It has been recommended as an efficient 
method for evaluating training intensity and recovery, and for 
improving the performance of endurance athletes.3-6 During 
incremental exercise, the lactate threshold (LT) is defined as 
the abrupt transition from slow increases to rapid exponential 
increases in blood lactate levels.7
The evaluation of lactate threshold in athletes has evolved, 
from the 4 mmol universal lactate threshold, to the more indi-
vidualized Onset of Blood Lactate Accumulation, and to the cur-
rent Maximal Lactate Steady State standard. This progression has 
been due to better understanding of the physiological processes 
of lactate production and clearance, and the role of lactate during 
prolonged and submaximal exercise.5,8-12
However, most published studies on lactate threshold have 
compared homogeneous groups of athletes with similar aero-
bic capacity, or have made regression analyses on these  data.13-16 
Comparisons between different methods on lactate thresh-
old acquisition also remain controversial in the literature.17-19 
To  our knowledge, there is no comparative study evaluat-
ing lactate threshold methods in both lowly and highly trained 
endurance athletes. 
OBJECTIVE
The purpose of this study was to evaluate different lactate thresh-
old methods, and determine which methods are most reliable 
for athletes with different physical conditioning and training 
programs. 
METHODS
This was an experimental laboratory study performed within 
the Sports Medicine Group of Faculdade de Medicina da 
Universidade de São Paulo. Twenty-seven male endurance run-
ners were recruited for this study from university campus run-
ning clubs. For the primary outcome (post-analysis method for 
the lactate threshold in the same group), the sample size was cal-
culated after a five-athlete pilot study, taking P < 0.05 and power 
= 80%. The sample size was estimated as 10 individuals per 
group. We added a minimum of 20% more subjects to account 
for potential data loss. 
The subjects were divided into two distinct groups based on 
the responses to a questionnaire: 15 highly trained runners (min-
imum of 4 training runs per week for 6 consecutive months, and 
a long-distance training pace less than or equal to 5.0 min/km) 
and 12 lowly trained runners (long-distance training pace 
greater than 5.0 min/km, with a maximum of 3 runs per week 
and a maximum of 6 consecutive training months). The exclu-
sion criteria were previous cardiorespiratory disease and 
musculoskeletal running-related injuries. No athlete was cur-
rently taking any medications. 
Oxygen consumption (VO2) was measured continuously 
and monitored by means of a breath-by-breath gas analyzer on a 
treadmill (h/p/cosmos, Pulsar, Germany) using a metabolic ana-
lyzer (CPX/D Med Graphics, St. Paul, USA)
The mean physiological characteristics of the highly trained 
group were: age 33.7 ± 10.3 years; training velocity: 4.0 ± 0.6 min/km; 
resting heart rate 68.7 ± 14.7 bpm; and VO2max: 52.4 ± 5.3 ml/kg/min. 
Characteristics of the lowly trained group were: age 37.2 ± 9.3 years; 
training velocity: 5.3 ± 0.9 min/km; rest heart rate 79.3 ± 15.2 bpm; 
and VO2max: 43.4 ± 5.7ml/kg/min. 
The Institutional Review Board approved this research and 
informed consent was obtained from each subject prior to partici-
pation. This research followed the Helsinki Declaration principles.20
Lactate protocol
A washout period of 24 hours with no physical activity was 
requested for all participants prior to the experiment. The sub-
jects then performed an incremental treadmill test to directly 
measure their lactate threshold. All subjects did the test at the 
same location, with the same equipment, and under similar ther-
mal  conditions (temperature 21-26°C, humidity 33-66%, baro-
metric pressure 688 mmHg). Throughout the protocol, treadmill 
elevation was kept constant at a 1% grade to duplicate the energy 
cost of over-ground running.21 
The subjects first performed a 3-minute warm-up run at 30% 
of their long-distance training velocity. At the beginning of the 
incremental test, the treadmill velocity was set at 70% of the esti-
mated long distance training velocity, depending on the running 
ability of each participant (it is known that performance in com-
petition is an appropriate criterion for valid laboratory tests).3,22 
Heart rate and Borg scale were recorded each minute. Stage 
length was set at 4 minutes,21 with running velocity increases of 
1 km/h per stage until volitional exhaustion was reached (as mea-
sured from the Borg scale). Fingerprint whole-blood samples were 
taken between the points of 3.5 and 4 minutes in each stage and 
were immediately analyzed in an automated blood-lactate ana-
lyzer (Accutrend Lactate, Typ3012522) without treadmill proto-
col interruption. Blood samples were collected for two additional 
stages following exponential inflection of the lactate point.
Calculating lactate threshold
The basis for determining the lactate threshold is that there is an 
inflection point at a given workload (i.e. running velocity) where 
blood lactate exponentially increases with a corresponding increase 
in workload.17,18,21,23,24 It is used to define the  highest work rate or O2 
uptake (oxygen consumption) at which athletes can maintain their 
efforts over a specified time frame.25 An individual blood-lactate 
Post-analysis methods for lactate threshold depend on training intensity and aerobic capacity in runners. An experimental laboratory study | ORIGINAL ARTICLE
Sao Paulo Med J. 2016; 134(3):193-8     195
profile was created for each subject by plotting running velocity 
(km/h) at each stage of the test (x-axis) versus blood-lactate con-
centration attained at each stage (y-axis).10,21,24,26,27
Three methods commonly cited in the literature were used to 
define the inflection point (Figure 1): 
1. Increase of 1 mmol/l blood lactate (DT1): the work rate that 
just precedes a rise in blood lactate concentration of > 1 mmol/l 
between two stages estimates the lactate threshold.10,21,26,27
2. Absolute value of 4 mmol/l blood lactate (4 mmol): work-
load when the concentration of lactate in the blood reaches 
4 mmol/l.10,21,26,27
3. Semi-log method (semi-log): based on a logarithmic scale 
(blood lactate) in which the exponential blood lactate curve 
is divided into two linear segments that cross each other; the 
point of intersection is the lactate threshold.17,18 
Statistical analysis
The normality curve was addressed by means of histograms and it 
was decided to use parametric tests. Baseline characteristics were 
analyzed first to demonstrate homogeneity. The threshold values of 
each method were compared with repeated-measurement analyses 
of variance (ANOVA). When a significant difference was attained, 
Tukey’s post-hoc test was performed. Statistical significance was 
denoted as P < 0.05 (STATA-9 for Windows).
RESULTS
Before analyzing the relationship between lactate threshold and 
velocity, the associations between lactate threshold and base-
line characteristics such as age, heart rate, VO2max and train-
ing regularity were assessed. This analysis showed that neither 
demographic nor baseline characteristics could explain associa-
tions with lactate threshold, except, logically, for the dependent 
variables of training regularity between groups and VO2max. 
As expected, the lactate thresholds of the highly trained 
group were obtained at higher velocity stages than those of the 
lowly trained group in all tested methods. When considering all 
subjects (both the highly trained and the lowly trained groups), 
comparison of lactate threshold methods showed significant dif-
ferences between the DT1 and 4 mmol methods, and between the 
semi-log and 4 mmol methods. There was no statistical difference 
between DT1 and semi-log (Figure 2).
10
1 mmol/l increase
method (DT1)
(a)
1 mmol/l increase
 threshold method
9
8
7
6
5
4
3
2
11
12
13
14
15
16
Velocity (km/h)
Lactate (mmol/l)
Log lactate (mmol/l)
17
18
19
20
21
1
(b)
semi-log method
(intersection)
0.9
0.8
0.7
0.6
0.5
11
12
13
14
15
16
Velocity (km/h)
17
18
19
20
21
Figure 1. Example of the three post-analysis methods for lactate threshold applied to one subject: (a) the velocity before the increase of 
1 mmol/l blood lactate (DT1); the velocity at which the blood lactate exceeds the value of 4 mmol/l (4 mmol); and (b) the velocity at the 
intersection of two interpolated lines on the semi-logarithmic scale (semi-log).
18
*#
16
14
12
10
8
6
4
DT1
semi-log
Velocity (km/h)
4 mmol
Figure 2. Box plot of the velocity of the lactate threshold of all subjects 
obtained using the DT1, semi-log and 4 mmol post-analysis methods.
*Significant difference between DT1 and 4 mmol methods (P < 0.05), and 
#significant difference between semi-log and 4 mmol (P < 0.05), ANOVA (analysis 
of variance), Bonferroni post-hoc test.
ORIGINAL ARTICLE | Fernandes TL, Nunes RSS, Abad CCC, Silva ACB, Souza LS, Silva PRS, Albuquerque C, Irigoyen MC, Hernandez AJ
196     Sao Paulo Med J. 2016; 134(3):193-8
When the groups were compared separately (highly trained 
and lowly trained), the 4 mmol measurement was found to be sig-
nificantly lower than the DT1 and semi-log measurements in the 
lowly trained group. The DT1 and semi-log measurements were 
not statistically different in this group (Figure 3). In the highly 
trained group, a significant difference was only found between 
the DT1 and the 4 mmol methods. 
DISCUSSION
The most important finding of this study was the differences in lactate 
threshold measurement methods between highly and lowly trained 
endurance runners. The method with fixed blood lactate of 4 mmol/l 
underestimated the lactate threshold in the lowly trained group. 
Sargent et  al.28 identified differences in lactate threshold 
between different groups of subjects, such as men versus women. 
On the other hand, Smekal et  al.29 reported that blood lactate 
concentration at the maximal lactate steady state was indepen-
dent of both endurance capacity and sex. Other authors have 
showed comparisons between trained and untrained individuals 
through using cardiorespiratory tests.30,31 One notable character-
istic of our study is that we only used male subjects and made 
comparisons between controlled training levels (high and low) 
instead of between trained and sedentary subjects.
One explanation for the different values of measurement 
methods is that error is introduced when the curves do not fol-
low the mathematical physiological functions.19 
Subjects with different performance levels often have differ-
ent mechanical running responses and consequently different 
metabolic demands.32 Our study also agreed with the literature 
regarding higher lactate threshold values in trained individuals. 
Kumagai et al.33 showed that aerobic training increased the lac-
tate threshold, with a concomitant improvement in both endur-
ance and middle-distance performance.
Individuals with greater endurance capacity have faster oxy-
gen kinetics.34 The higher values of lactate thresholds in the highly 
trained subjects may reflect more efficient peripheral and central 
exchange during exercise.34 During low-intensity exercise, blood 
lactate formation and removal depends on the intracellular/tissue 
balance among the glycolytic (cytosol) and oxidative (mitochon-
dria) processes.35 It seems that in trained individuals, these vari-
ables are more predictable and have controlled behavior. 
Joyner et al.36 suggested that running performance could be 
explained by VO2max, running economy and fractional utiliza-
tion of VO2max. Moreover, they suggested that the lactate thresh-
old  integrates all three of these variables and is the best physio-
logical predictor of distance running performance1, given that it 
is detectable in both trained and untrained individuals.10
It is known that the average value for the lactate threshold 
in normal subjects is 3.7 mmol and that serum blood lactate at 
the lactate threshold is not equal for all individuals (range: 1.5 to 
7.5 mmol) and also changes in a single individual.37 Although the 
4 mmol lactate protocol is an easy method for estimating lactate 
threshold, the fixed value of 4.0 mmol does not take these physi-
ological conditions into consideration and may underestimate lac-
tate threshold, as shown in this study.38 
The clinical relevance of this study relates to the populations 
tested. Most people are not competitive endurance athletes, yet 
still need predictions of aerobic threshold and exercise prescrip-
tions for health issues. Our results suggest that lowly trained sub-
jects would benefit from semi-log or DT1 lactate threshold meth-
ods in clinical practice. 
The main limitation of this study relates to the treadmill pro-
tocol, such as the stage duration and initial running velocity. 
Due to the large variation of treadmill protocols in the  lactate 
threshold literature, direct comparisons of our results with pre-
vious studies may not be appropriate. Despite training group 
characteristics that were very specific (frequency, intensity and 
duration of training), we believe that they represent objective 
inclusion criteria and, because of that, the results may be repro-
ducible. Future studies should examine lactate threshold meth-
ods and cardiorespiratory performance in both highly trained 
and lowly trained groups. We suggest that these groups should be 
stratified according to training frequency, intensity and duration.
CONCLUSION
The 4 mmol protocol did not show lactate threshold measure-
ments comparable with with DT1 and semi-log protocols among 
lowly trained athletes. 
18
*#
*
16
14
12
10
8
6
4
DT1
semi-log
highly trained
lowly trained
4 mmol
DT1
semi-log 4 mmol
Velocity (km/h)
Figure 3. Box plot of the velocities at the lactate threshold obtained 
using the DT1, semi-log and 4 mmol post-analysis methods in each 
group (highly trained and lowly trained). 
*Significant difference between DT1 and 4 mmol methods (P < 0.05), and 
#significant difference between semi-log and 4 mmol (P < 0.05), using ANOVA 
(analysis of variance) and the Bonferroni post-hoc test.
Post-analysis methods for lactate threshold depend on training intensity and aerobic capacity in runners. An experimental laboratory study | ORIGINAL ARTICLE
Sao Paulo Med J. 2016; 134(3):193-8     197
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Acknowledgements: The authors would like to thank Sean J. Driscoll for 
proofreading this manuscript
Sources of funding: Fundação de Amparo à Pesquisa do Estado de São 
Paulo (Fapesp) – Protocolo 2010/19631-2
Conflict of interest: None
Date of first submission: May 11, 2014
Last received: November 19, 2014 
Accepted: December 15, 2014
Address for correspondence: Tiago Lazzaretti Fernandes  
Laboratório do Estudo do Movimento  
Instituto de Ortopedia e Traumatologia, Hospital das Clínicas  
Faculdade de Medicina da Universidade de São Paulo 
Dr. Ovídio Pires de Campos, 333 — 2o andar  
São Paulo (SP) — Brasil 
CEP 05403-010 
Tel. (+55 11) 2661-6486 
E-mail: tiago.lazzaretti@usp.br

Post-analysis methods for lactate threshold depend on training intensity and aerobic capacity in runners. An experimental laboratory study.
Fernandes, Tiago Lazzaretti,Nunes, Rômulo Dos Santos Sobreira,Abad, Cesar Cavinato Cal,Silva, Andrea Clemente Baptista,Souza, Larissa Silva,Silva, Paulo Roberto Santos,Albuquerque, Cyro,Irigoyen, Maria Cláudia,Hernandez, Arnaldo José