Biomarkers of physical activity and exercise

Abstract

Traditionally, biomarkers have been of interest in sports in order to measure performance, progress in training and for identifying overtraining. During the last years, growing interest is set on biomarkers aiming at evaluating health-related aspects which can be modulated by regular physical activity and sport. The value or concentration of a biomarker depends on many factors, as the training status of the subject, the degree of fatigue and the type, intensity and duration of exercise, apart from age and sex. Most of the biomarkers are measured in blood, urine and saliva. One of the main limitations for biochemical biomarkers is that reference values for blood concentration of biomarkers specifically adapted to physically active people and athletes are lacking. Concentrations can differ widely from normal reference ranges. Therefore, it is important to adapt reference values as much as possible and to control each subject regularly, in order to establish his/her own reference scale. Other useful biomarkers are body composition (specifically muscle mass, fat mass, weight), physical fitness (cardiovascular capacity, strength, agility, flexibility), heart rate and blood pressure. Depending on the aim, one or several biomarkers should be measured. It may differ if it is for research purpose, for the follow up of training or to prevent risks. For this review, we will get deeper into the biomarkers used to identify the degree of physical fitness, chronic stress, overtraining, cardiovascular risk, oxidative stress and inflammation.
Copyright AULA MEDICA EDICIONES 2015. Published by AULA MEDICA. All rights reserved.

Full text

237
Nutr Hosp. 2015;31(Supl. 3):237-244
ISSN 0212-1611 • CODEN NUHOEQ
S.V.R. 318
Biomarkers of physical activity and exercise
Gonzalo Palacios 1,2, Raquel Pedrero-Chamizo1, Nieves Palacios3, Beatriz Maroto-Sánchez1,
Susana Aznar4 and Marcela González-Gross1,2 on behalf of EXERNET Study Group
1ImFINE Research Group. Technical University of Madrid. Spain. 2CIBERobn (Fisiopatología de la Obesidad y la Nutrición
CB12/03/30038), Instituto de Salud Carlos III, Spain. 3Sport Medicine Center. High Sports Council. Spain. 4PAFS Research
Group. University of Castilla-La Mancha. Spain.
Abstract
Traditionally, biomarkers have been of interest in
sports in order to measure performance, progress in tra-
ining and for identifying overtraining. During the last
years, growing interest is set on biomarkers aiming at
evaluating health-related aspects which can be modula-
ted by regular physical activity and sport. The value or
concentration of a biomarker depends on many factors,
as the training status of the subject, the degree of fatigue
and the type, intensity and duration of exercise, apart
from age and sex. Most of the biomarkers are measured
in blood, urine and saliva. One of the main limitations
for biochemical biomarkers is that reference values for
blood concentration of biomarkers specifically adapted
to physically active people and athletes are lacking. Con-
centrations can differ widely from normal reference ran-
ges. Therefore, it is important to adapt reference values
as much as possible and to control each subject regularly,
in order to establish his/her own reference scale.
Other useful biomarkers are body composition (spe-
cifically muscle mass, fat mass, weight), physical fitness
(cardiovascular capacity, strength, agility, flexibility),
heart rate and blood pressure. Depending on the aim, one
or several biomarkers should be measured. It may differ
if it is for research purpose, for the follow up of training
or to prevent risks. For this review, we will get deeper
into the biomarkers used to identify the degree of phy-
sical fitness, chronic stress, overtraining, cardiovascular
risk, oxidative stress and inflammation.
(Nutr Hosp 2015;31(Supl. 3):237-244)
DOI:10.3305/nh.2015.31.sup3.8771
Key words: Physical fitness. Health. Performance. Bio-
marker. Cortisol.
BIOMARCADORES DE LA ACTIVIDAD FÍSICA
Y DEL DEPORTE
Resumen
Tradicionalmente, los biomarcadores han sido de inte-
rés en las ciencias del deporte para medir el rendimien-
to, el progreso en el entrenamiento y para identificar el
sobreentrenamiento. Durante los últimos años, cada vez
hay mayor interés en evaluar los efectos relacionados con
la salud que se producen en el organismo debidos a una
actividad física regular y al deporte. El valor o la concen-
tración de un biomarcador depende de muchos factores,
como el grado de entrenamiento, el grado de fatiga y del
tipo, la intensidad y la duración del ejercicio, aparte de
la edad y del sexo. La mayor parte de los biomarcadores
se miden en sangre, orina y saliva. Una de las principales
limitaciones que presentan los biomarcadores bioquími-
cos es la falta de valores de referencia adaptados específi-
camente para deportistas y personas físicamente activas.
Las concentraciones pueden variar considerablemente
de los valores de referencia normales. Por lo tanto, es
importante adaptar los valores de referencia siempre y
cuando sea posible y controlar a cada sujeto regularmen-
te, con el fin de establecer su propia escala de referencia.
Otros biomarcadores útiles son la composición corpo-
ral (específicamente masa muscular, masa grasa, peso),
la condición física (capacidad cardiorrespiratoria, fuer-
za, agilidad, flexibilidad), frecuencia cardíaca y presión
arterial. Dependiendo de la finalidad, será conveniente
analizar uno o varios biomarcadores. Para esta revisión,
profundizaremos en los biomarcadores que se emplean
para evaluar condición física, fatiga crónica, sobreentre-
namiento, riesgo cardiovascular, estrés oxidativo e infla-
mación.
(Nutr Hosp 2015;31(Supl. 3):237-244)
DOI:10.3305/nh.2015.31.sup3.8771
Palabras clave: Condición física. Salud. Rendimiento.
Biomarcador. Cortisol.
Correspondence: Marcela González-Gross.
ImFINE Research Group.
Department of Health and Human Performance.
Facultad de Ciencias de la Actividad Física y del Deporte-INEF.
Universidad Politécnica de Madrid.
C/ Martín Fierro 7. E.
28040 Madrid.
E-mail: marcela.gonzalez.gross@upm.es
Background
A biomarker (biological marker) is a measurable
product or substance used as an indicator of the biolo-
gical state, to objectively determine the body’s physio-
logical or pathological processes. In sport, biomarkers
are key parameters to assess the impact of exercise on
different systems, tissues and organs1. Therefore, we
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ENERGY EXPENDITURE AND PHYSICAL ACTIVITY: METHODOLOGICAL ISSUES
can estimate parameters for assessing the degree of fit-
ness, muscle damage, hydration/dehydration, inflam-
mation, oxidative damage, fatigue, overtraining, etc,
which facilitate the evaluation of the response of the
human body at the different levels of physical activity
or training being carried out. Biomarkers can be used
to measure the impact of training on the long term or
the acute effect of exercise. The value or concentra-
tion of a biomarker depends on many factors, as the
training status of the subject, the degree of fatigue and
the type and duration of exercise, apart from age and
gender, among others. Climate can also play a role,
mainly temperature, humidity and wind speed. Exer-
cise can be classified according to the duration as the
following: around 20s (demand for anaerobic energy
up to 90%), exercise lasting 20s to 1 min (aerobic and
anaerobic energy) or exercises that extend over 1 min
(aerobic energy more than 50%). Intensity is also an
influencing factor on biomarker concentration. Most
of the biomarkers are measured in blood, urine and
saliva. In elite sports, non-invasive samples like urine
and saliva have a preference. Other useful biomarkers
are body composition (specifically muscle mass, fat
mass, weight), physical fitness (cardiovascular capa-
city, strength, agility, flexibility), heart rate and blood
pressure.
Depending on the aim, one or several biomarkers
should be measured. It may differ if it is for research
purpose or for the follow up of training.
Interest
Traditionally, biomarkers have been of interest in
sports in order to measure performance, progress in
training and for identifying overtraining2. During the
last years, growing interest is set on biomarkers aiming
at evaluating health-related aspects which can be mo-
dulated by regular physical activity and sport3. Addi-
tionally, as promotion of physical activity is supported
by most of public health authorities, the evaluation of
the biological response to exercise is also a need in
the amateur athlete. For this review, we will get deeper
into the biomarkers used to identify the degree of phy-
sical fitness, chronic stress, overtraining, cardiovascu-
lar risk, oxidative stress and inflammation.
Controversy
Controversy exists regarding if biochemical bio-
markers are really useful for the monitoring of training
progress and adaptation, and some trainers do not in-
clude biomarkers in their season planning2. Less con-
troversy exists in regard to identifying risk situation,
like overtraining, nutrient deficiencies, etc. Unfortu-
nately, there is no gold standard for monitoring most
of the processes; therefore, the analysis of several bio-
markers is recommended.
Limitations
Reference values for blood concentration of biomar-
kers specifically adapted to physically active people
and athletes are lacking. Therefore, for most of the bio-
markers measured routinely in the laboratory, reagent
manufactures’ reference values are used. In the authors’
opinion, this can lead to misclassification or wrong
interpretation of the results. Our research group is cu-
rrently working on reference values specifically adapted
to athletes of different sports (E. Diaz, non-published
data). It is important to bear in mind that highly trained
people can have concentrations of biomarkers which
would be pathological in non-trained people, even in
routine hematology and biochemistry parameter. There-
fore, it is important to adapt reference values as much as
possible and to control each subject regularly, in order
to establish his/her own reference scale.
Current state and perspective
Markers of Physical fitness
Physical fitness is a set of attributes that people have
or achieve and it is referred to the capacity of a person
to meet the varied physical demands of their activities
of daily living and/or sport practice without experimen-
ting fatigue. Physical fitness is not only a predictor of
morbidity and mortality for cardiovascular disease4. It is
nowadays considered one of the most important health
markers because it integrates most of the body functions
(skeletomuscular, cardiorespiratory, hematocirculatory,
psychoneurological and endocrine-metabolic) invol-
ved in the performance of daily physical activity and/or
physical exercise3. Accordingly, when physical fitness
is tested, the functional status of all these systems is ac-
tually being checked. Physical fitness is in part geneti-
cally determined, but it can also be greatly influenced
by environmental factors, such as physical exercise, se-
dentary habits, harmful lifestyles, etc.
Physical fitness components can be differentiated
between a health-related and a performance-related
approach that pertain more to athletic ability, being
the health-related components more important to pu-
blic health than are the components related to athletic
ability5. According to Bouchard et al.1, the health-re-
lated fitness components of a person include a cardio-
respiratory component (e.g. maximal aerobic power or
heart function); a muscular component (e.g. strength,
power or muscular endurance); a motor component
(e.g. agility, balance and co-ordination); a morpholo-
gical component (e.g. body composition, bone density
or flexibility); and a metabolic component (e.g. glu-
cose tolerance, lipid and lipoprotein metabolism and
substrate oxidation characteristics)6. There are nume-
rous tests for measuring physical fitness, ranging from
self-assessment techniques over simple field test to
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Biomarkers of physical activity and exercise
239
more sophisticated laboratory tests. One may choose
to employ a different measure depending on the speci-
fic objectives of the investigation and cost constraints.
The World Health Organization (WHO), considered
the maximal oxygen consumption (VO2max) as the
single best indicator of cardiorespiratory fitness7 and
it can be estimated using a maximal or sub-maximal
test (e.g. treadmill or bicycle tests, 2 km. walk test,
20-m shuttle run, 6 min. walk test). According to mus-
cular component, the handgrip strength test is one
of the most used tests for assessing muscular fitness
being a strong predictor of morbidity and mortality8.
For assessing power strength or muscular endurance
jump, dynamic sit-up and bent-arm hand tests have
been used with young, adult and older people. Agili-
ty, balance, speed or co-ordination is included in the
motor component. Agility is a combination of speed,
balance, power and coordination3. Some tests used to
measure motor component are 30-m sprint test and 4 x
10-m shuttle run test for young people and 30-m walk
test and 8-foot-and-go, for older adults. For measuring
static balance, single leg balance, with or without open
eyes, is a good alternative test. Flexibility is a morpho-
logical component; chair sit-and-reach test and back
scratch test are two validated tests for measuring this
capacity (Fig. 1).
Fig. 1.—Some tests used
for the evaluation of phy-
sical fitness in elderly
(modified from 9).
Test item Assessment category Description
One leg []
Static balance
Number of seconds during which the
participant kept balance on one leg.
The maximum time allowed for the test
was 60 s.
Chair stand
Lower body s strength Number of full stands in 30 seconds with
arms folded across chest.
Arm curl
Upper body strength Number of biceps curls in 30 seconds
holding hand weight.
Chair sit -and -reach
Lower body flexibility
From sitting position at front of chair, with
leg extended and hands reaching toward
toes, number of centimetres from extended
fingers to tip of toe.
Back scratch
Upper body flexibility With one hand reaching over shoulder and
one up middle of back, number of centime-
tres between extended middle fingers.
8 -foot -and -go
Agility/Dynamic balance Number of seconds required to rise from
seated position, walk 2.45 metres, turn,
and return to seated position on chair.
30 -m walk
Walking speed Number of seconds required to walk
30metres.
6 -minute walk
Aerobic capacity Number of meters that can be walked in
6minutes around a 46 meters course.
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ENERGY EXPENDITURE AND PHYSICAL ACTIVITY: METHODOLOGICAL ISSUES
For use in clinical practice, reference values for men
and women in all age groups are needed. Some of the
most important studies that have provided reference
values are AVENA and HELENA3 studies, for Spanish
and European adolescents, respectively, and the senior
fitness test by Rikli & Jones and the EXERNET study9,
for Americans and Spanish elderly people, respectively.
Markers of chronic stress and fatigue
Cortisol
Cortisol is a steroid hormone synthesized from choles-
terol by enzymes of the cytochrome P450 located at adre-
nal cortex. It’s expressed following a circadian rhythm:
at midnight, cortisol blood levels are very low (someti-
mes even undetectable) and they increase overnight to
reach a peak in the morning. This rhythm is regulated
by the main circadian oscillator in the suprachiasmatic
nucleus which is located in the hypothalamus10. Cortisol
counters insulin effect by promoting high blood glucose
levels via stimulation of gluconeogenesis, the metabolic
pathway that synthesizes glucose from oxaloacetate. The
presence of cortisol triggers the expression of enzymes
critical for gluconeogenesis, facilitating this increase in
glucose production. Conversely, it also stimulates gly-
cogen synthesis in the liver, which decreases net blood
sugar levels. Thus, cortisol carefully regulates the level
of glucose circulating through the bloodstream: when
blood glucose has been depleted (for example during
fasting), cortisol ensures a glucose basal concentration
by activating gluconeogenesis11.
Cortisol shows other metabolic functions. Among
others, it allows a correct pH regulation of extracellular
liquid: when cells loose too much sodium, it accelerates
the rate of potassium excretion. Therefore, cortisol regu-
lates the action of cellular sodium-potassium pumps to
reach an ion equilibrium after a destabilizing event11. Cor-
tisol’s weakening effects on the immune response have
also been well documented. T-lymphocyte cells (T-cells)
are activated by cytokine molecules (interleukins) via a
signaling pathway. Cortisol prevents specific T-cells re-
ceptors to recognize interleukin signals and reducing pro-
liferation of T-cells, which provokes a decrease of inflam-
mation course. In the same way, it reduces inflammation
due to inhibition of histamine secretion. Cortisol’s ability
to prevent the immune response can render people who
suffer from chronic stress highly vulnerable to infection12.
While it is important for adrenal glands to secret
more cortisol in response to psychological or physical
stress, it is also fundamental that cortisol levels return
to normal values following a stressful event. Unfortu-
nately, in some athletes the stress response to an inten-
sive exercise is activated so often that the metabolic
pathways do not always have a chance to return to a
normal situation. This can lead to health problems,
resulting, among others, in chronic stress and fatigue.
Training load as measured by the session-RPE (Ra-
ting of Perceived Exertion) is a subjective method of
quantifying the load placed on an athlete. Measured ses-
sion-RPE in 8 young elite middle-distance runners for
8 weeks, showed that this indicator of training load was
able to detect states of overreaching13. In another way,
measurement of the countermovement jump (CMJ) sco-
re can be used as an indicator of neuromuscular perfor-
mance and therefore it has been used to assess fatigue in
different kinds of athletes. Finally, salivary cortisol co-
rrelates with both physical magnitudes14. Thus, the me-
asurement of session-RPE, CMJ score and salivary cor-
tisol is used to monitor the training process in different
kinds of athletes. Post-exercise salivary cortisol respon-
ses were significantly different depending on the intensi-
ty. For example, immediately after a high intensity acute
resistance exercise, salivary cortisol showed a significant
elevation of 97% from baseline values, while there was
no difference when the intensity of exercise was very
low15. In addition to the intensity, another factor that may
affect the salivary cortisol response is the training status
of the subjects16. Highly-trained strength athletes show
an inverse and significant correlation with neuromus-
cular performance. Kraemer et al. 17 through their study
on cortisol and performance of a group of highly-trai-
ned soccer players throughout a season, concluded that
athletes starting the season with elevated cortisol values
may experience significant reductions on performance
during the season. Similar results have been obtained
with middle and long distance runners18. Individuals pre-
senting higher long-term salivary cortisol levels showed
a significant tendency to be those with lower CMJ scores
throughout the season. However, when correlation was
studied in a shorter period of time, a significant positive
trend was observed between runners with higher weekly
salivary cortisol concentrations with higher CMJ scores.
Further studies are needed to explain these opposite ten-
dencies depending of measure time.
Testosterone
This steroid hormone belonging to the group of an-
drogens in the body facilitates increased muscle mass
and strength, increased combativeness and aggression
of athletes and allows greater reduction of muscle fat.
Reference ranges are 300 - 1000 ng/dL for men and
15-70 ng/dL for women. A disproportionate rise in the
physiological stress response induces an increase in
cortisol secretion which could in turn inhibit the syn-
thesis of testosterone. The cortisol/testosterone ratio is
an index used to measure chronic fatigue in athletes16.
Markers of overtraining
Lactate
Muscles always produce lactate, even at rest (0.8 –
1.5 mmol/L), but lactate increases incrementally with
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Biomarkers of physical activity and exercise
241
exercise intensity. At a certain intensity lactate increases
exponentially, this is called the lactate threshold, which
occurs on average at a blood lactate concentration of 4.0
mmol/L. Fatigue onset appears fast above the lactate
threshold limit, while efforts just below this limit can
be sustained for hours when athletes are well trained.
This training allows also raising the lactate threshold
to the highest genetic potential of each subject. Howe-
ver, to perform too much training at or above the lactate
threshold can result in overtraining. Thus, blood lactate
measurement is used to determine not only the lactate
threshold, but also the correct intensity of the exerci-
se and the time needed for recovery. Lactate testing is
used all over the world by researchers and athletic coa-
ches. It can be considered as the current gold standard
for determining exercise intensity and for determining
whether or not training is producing the desired physio-
logical effect. Briefly, muscle contraction starts on an
electrical impulse from the brain, which is transmitted
to muscle cells by means of the acetylcholine liberated
at the motor neuron synapses. This produces a change
in the membrane potential due to the leak out of potas-
sium ions to the extracellular space, allowing calcium
ions to be released from the endoplasmic reticulum and
finally to trigger contraction of the muscle fiber. But
during high-intensity or long-time exercises, potassium
ions continuously leak out of the muscle cell into the
extracellular space, causing a depolarizing effect in the
membrane, as the charge difference between inside and
outside cell decreases. As a consequence, electrical cu-
rrents have a harder time getting in and muscle contrac-
tions become weaker. Recent studies have shown that
far from causing fatigue in the exercising muscle, lacta-
te production actually prevents fatigue by counteracting
the effects of depolarization produced by the potassium
ions outflow19.
Creatine (phospho) kinase (CK or CPK)
CK is used as a marker of muscle fiber damage.
Blood concentrations increase with increasing exerci-
se intensity and duration. There is an adaptation due to
training, enabling the levels in trained people to rise
less than in sedentary people. Elevated baseline values
indicate trauma or overtraining and its concentration
can be used to monitor activity around athletes who
have got a muscle injury20 (Table I).
Creatinine
This metabolite is an end product of muscle meta-
bolism. It originates from muscle creatine degradation
which in turn is produced by hydrolysis of creatine
phosphate, by the action of creatine phosphate kinase
(CPK). Creatinine clearance in the human body occurs
almost exclusively by glomerular filtration, which is
an important indicator of renal function. Renal excre-
tion, unlike urea, does not depend on diuresis. Concen-
trations are substantially constant in each individual
independent of diet, muscle mass being the main de-
terminant. Commonly, creatinine is measured to assess
whether renal function is adequate or not. In sports
medicine, creatinine is typically used to assess the
overall health of the athlete but normal values based
on the normal population are not adequate for athletes.
Normal references range from 0.7-1.3 mg/dl in adult
men. In athletes, levels are usually high, depending on
training, time of the season, which can induce changes
in creatinine levels due to changes in the homeostasis
of the body, which can lead to errors in biochemical
and hematological parameters2. No specific referen-
ces have been defined for athletes. Therefore, most
commonly elevated creatinine is an indicator of a high
degree of training or overtraining rather than a case
of renal pathology. Creatinine concentration should be
taken with caution, as it can be up to 1.4 mg/dl without
suffering from renal disease. The interpretation of
creatinine values should be done individually, taking
into account gender, age and weight of the athlete.
Ammonia
In athletes, the accumulation of ammonia in the blood
is dependant on the effort intensity. During physical
exercise, the two main mechanisms by which ammonia
accumulates are resynthesis of ATP from the breakdown
of phosphocreatine (PC) and the deamination of amino
acids. Rising ammonia is related to fast twitch muscle
fibers. Therefore, the analysis of the values of ammo-
nia can serve both as a marker for this type of exercise
and as a marker of intense muscular effort fibers. The
normal range of ammonia is 15 to 45 µg/dL. Elevated
blood ammonia levels indicate a physiological response
in sprinters (purely anaerobic metabolism), while lower
rates correspond to medium or long distance runners
(predominantly aerobic metabolism)21.
Lactate dehydrogenase (LDH)
LDH is a catalytic enzyme found in most tissues
of the body, and specifically in heart, liver, kidneys,
muscles, blood cells, brain and lungs. LDH plays an
important role in anaerobic energy metabolism, redu-
Table I
Training status depending on creatine kinase
concentrations
CK concentration Interpretation
200UI Training adaptation
200-250UI Elevated training levels
>300UI Possible overtraining and muscle damage
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ENERGY EXPENDITURE AND PHYSICAL ACTIVITY: METHODOLOGICAL ISSUES
cing pyruvate to lactate at the end of the glycolysis.
When there is muscle damage or muscle fiber destruc-
tion, LDH serum levels are significantly increased. In
addition, LDH has a variety of isoenzymes, which are
specific to different tissues, which provide additional
information on the origin of muscle damage20.
Uric acid
Uric acid is the end product of purine metabolism,
which increases after intense exercise. Its concentra-
tion should be stable during the competition season.
Elevated levels may be due to intense training, to high
energy demands and small muscle damage as a result
of overtraining. The increase may also be related to
intake of purine-rich foods and food supplements, and
body weight changes in athletes2.
Urea
Urea is mostly formed in the liver as the waste pro-
duct from the breakdown of proteins (amino acids).
Normal blood urea concentrations for optimum trai-
ning loads are 5-7 mmol/L. Very long training sessions
cause an increase in the urea concentration in blood,
liver, skeletal muscles, urine and sweat. It is used as a
marker of protein catabolism. Thus, the measurement
of urea allows to evaluate the degree of protein utili-
zation as an energy substrate, the degree of effort in a
competitive test session in particular, and the level of
athlete’s overtraining21.
Markers of cardiovascular risk
Homocysteine
Homocysteine (Hcy) is an intermediate sulfhy-
dryl-containing amino acid derived from methioni-
ne. In the methionine cycle, methionine converses to
S-adenosyl methionine and to Hcy. Hyperhomocystei-
nemia (high Hcy blood levels) can be classified depen-
ding of total serum plasma Hcy concentration in three
stages: moderate (for concentrations between 16 and
30 µM/L), intermediate (31–100 µM/L), and severe
(for concentrations higher than 100 µM/L). Hyperho-
mocysteinemia can be the result of disturbed Hcy me-
tabolism, principally when deficiencies in folic acid,
vitamin B6, or vitamin B12 are present, as these vita-
mins are coenzymes of several regulating enzymes22.
Hyperhomocysteinemia can be also caused by other
factors independent of diet like genetic disorders in
methionine and homocysteine metabolism, including
mutations in cystathione b-synthase, methionine syn-
thase and methylenetetrahydrofolate reductase (MTH-
FR)21. Increased Hcy levels are associated with several
disorders, like cardio and cerebrovascular diseases22
and neurodegenerative diseases that affect the central
nervous system, such as epilepsy, stroke, Alzheimer’s
disease and dementia23.
There are several proposed mechanisms to exp-
lain the toxicity of Hcy. For this review, we will only
describe the two main ones. The first one is related
to oxidative stress. Oxidation of the thiol terminal
group of Hcy, when Hcy binds proteins (by forming
a disulphide bridge), low-molecular plasma thiols or a
second Hcy molecule, produce an increase of produc-
tion of reactive species. Those free radicals induce the
subsequent oxidation of proteins, lipids and nucleic
acids24 and can lead to the endothelial dysfunction and
damage to the vessel wall, followed by platelet acti-
vation and thrombus formation. Homocysteinylation
represent the second main mechanism of Hcy toxicity
and it consists on modification of protein structure due
to disulphide bond. Degree of the protein homocys-
teinylation increases with increased plasma Hcy and
causes immune activation, autoimmune inflammatory
response, cellular toxicity, cell death and enhanced
protein degradation22.
Because physical activity contributes to reduce car-
diovascular risk factors and Hcy is one of such fac-
tors, theoretically Hcy could be used as biomarker of
cardiovascular health when physical activity is perfor-
med. However, results obtained from several studies
are contradictory and sometimes inconclusive, may
be due to different kind of exercise, intensities, dura-
tion, with or without prior training, etc. A recent study
carried-out by Iglesias-Gutierrez et al.25 about serum
Hcy concentration during an acute bout of exercise
showed an increase at the beginning of exercise and
a subsequent decrease at the end, the basal value be-
ing recovered 19 hours post-exercise. Due to this long
period to reach again the initial Hcy levels, differen-
ces in timing of post-exercise sample collection could
explain these discrepancies on Hcy levels variations
after exercise described in the literature. An important
point to be considered is the maximum Hcy concentra-
tion reached during exercise and how long high Hcy
concentration persists in blood. In this study, authors
did not observed any concentration above 15 µmol/L,
the upper limit of Hcy normal range, beyond which
it falls in hyperhomocysteinemia. However, this does
not mean that the transient increase observed in Hcy
is lacking of physiological relevance. A meta-analysis
has shown that an average Hcy increase of 1.9 µmol/L
was associated to a 16% higher risk of isquemic heart
disease26. Nevertheless, this increase in cardiovascular
disease risk observed by Wald et al.26 refers to a sustai-
ned elevation of Hcy throughout lifespan, while Igle-
sias-Gutierrez et al.25 refers to an elevation as response
of an acute exercise.
In a recent review on effects of physical activity
on Hcy levels, authors underscored that a high daily
physical activity can help to control Hcy levels and
thus reducing cardiovascular disease risk27. However,
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Biomarkers of physical activity and exercise
243
an intensive and acute exercise tends to increase Hcy
blood levels28. The effect of aerobic training is more
controversial: resistance training seems to reduce Hcy
levels while intensive training increases them. Authors
suggest to carry-out further studies to study changes
in homocysteine induced by combined exercise pro-
grams (i.e., aerobic and resistance).
Cardiac troponin
Cardiac troponin consists of two protein complexes
(cTnI and cTnT) which regulate muscle contractile
function. They are present in skeletal and cardiac mus-
cle. Increased concentration of the cardiac isoforms
(TnI and TnT) indicates that there has been muscular
heart damage. Therefore, both markers are useful para-
meters to assess a cardiac event. However, the increa-
se after intense or prolonged exercise in the absence
of cardiac symptoms, suggests muscle lesions, due to
adaptation of training.
Markers of oxidative stress
Malondialdehyde (MDA) and protein carbonyls (PC)
Malondialdehyde (MDA) is a marker of oxidative
degradation of the cell membrane caused by lipid pe-
roxidation of unsaturated fatty acids. Protein carbon-
yls (PC) come from the oxidation of albumin or other
serum proteins and are used as a marker of oxidative
damage of proteins. Reference limits for PC are 0.30
to 0.36 nmol/mg29. PC and MDA are lower in trained
individuals. An increase can be due to stress caused by
increasing training loads. However, after adaptation to
training, concentrations decrease and return to normal
values.
Superoxide dismutase (SOD) and glutathione
peroxidase (GSH)
These are antioxidant enzymes modulated by phy-
sical activity. Resistance training increases moderately
enzymes activity29.
Reactive oxygen species (ROS)
There is growing evidence that the continued pre-
sence of high concentrations of free radicals is capa-
ble of inducing antioxidant enzymes and other defen-
se mechanisms. In this context, free radicals can be
viewed as beneficial rather than as harmful, since they
act as signals to improve the defenses when cells are
exposed to high levels of ROS. This is mainly due to
the regulation of endogenous antioxidant enzymes
such as glutathione peroxidase, manganese superoxide
dismutase (MnSOD), and γ-glutamylcysteine synthe-
tase. Therefore, we could state that training increases
the expression of antioxidant enzymes that in turn keep
decreasing ROS levels. The stimulated high levels of
ROS create more antioxidant enzymes, which do not
contribute to the oxidation of the body’s cells29.
Markers of inflammation
C-reactive protein
C-reactive protein (CRP) originates in the liver.
There are many stimuli that can cause an increase in
CRP concentrations, such as infection, trauma, sur-
gery, chronic inflammatory conditions, etc. In the
field of sports, intense physical activity can cause an
increase in CRP. However, continuous training causes
a reduction of CRP levels compared to baseline. This
is due to different mechanisms and processes taking
place while the body is adapting to training (improved
endothelial function, reduced inflammatory cytokine
production, antioxidant effects, increased insulin sen-
sitivity, etc). A higher CRP level after training indica-
tes lack of adaptation or overtraining, probably due to
oxidative stress (inflammation). However, after adap-
tation to training, values are normalized30.
Interleukin-6
Interleukin-6 (IL-6) is considered an anti-inflam-
matory cytokine that regulates acute inflammatory res-
ponse. Receptors are located in adipose tissue, skeletal
muscle and liver. IL-6 increases lipolysis in adipose
tissue and improves insulin sensitivity in the liver,
increases glycogenolysis in skeletal muscle. Intense
exercise training increases plasma concentrations of
IL-6 up to 100 times, indicating the beneficial effect of
physical exercise30.
Leukocytes
The white blood cells are part of the immune system,
produced in the bone marrow and lymphoid tissue. Af-
ter being synthesized they are transported by the blood
to the different parts of the body. The fundamental va-
lue of leukocytes is that they are specifically transpor-
ted into areas where there is inflammation, to provide
rapid and vigorous defense against any possible infec-
tious agent. Exercise causes a transient leukocytosis,
which magnitude is directly related to the intensity
of the exercise: it is more pronounced in response to
maximal exercise, and in untrained or poorly trained
individuals. An increased leukocyte value due to exer-
cise reaches again normal values within 24 hours.
026_Biomarcadores de la actividad física_Marcela González .indd 243 12/02/15 14:25

244
ENERGY EXPENDITURE AND PHYSICAL ACTIVITY: METHODOLOGICAL ISSUES
Conclusions
Biomarkers are useful tools for assessing and monito-
ring health, training status and performance. As there is
some controversy in the literature, depending on the pro-
cess to be monitored, a combination of biomarkers could
be useful. However, controversy exists regarding which
parameters are most relevant for monitoring fatigue. The
most researched and applied to muscle fatigue are cor-
tisol, lactate and IL-6. Also gaining increasingly more
importance is the measurement of ammonia, leukocytes
and oxidative stress parameters. The biomarkers of
muscle fatigue could be a prognostic tool to identifying
subjects who are at increased risk of poor adaptation to
training. Exercise, in particular, has a major influence on
the most widely used inflammatory biomarkers, inclu-
ding C-reactive protein and interleukin-6. Additionally
to biochemical biomarkers, the measurement of physi-
cal fitness components should be included in order to
monitor progress and adaptation to training, as fitness is
considered one of the most important markers of health.
Acknowledgements
The EXERNET Study Group (Research Network on
Exercise and Health in Special Populations) has been
supported by the Ministerio de Educacion y Ciencia
(DEP2005-00046/ACTI).
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