Effects of whole-body cryotherapy on serum mediators of inﬂammation
and serum muscle enzymes in athletes
, Gianluca Melegati
, Alessandra Barassi
, Giada Dogliotti
Gianvico Melzi d’Eril
, Benoit Dugue
, Massimiliano M. Corsi
Istituto Ortopedico R. Galeazzi, IRCCS, 20161 Milan, Italy
Department of Health Technology, University of Milan, Milan, Italy
Italian Rugby Federation, Italy
Department of Medicine and Surgery, San Paolo Hospital, University of Milan, Milan, Italy
Laboratory of Clinical Pathology, Institute of General Pathology, Medical Faculty, University of Milan, 20133 Milan, Italy
Laboratoire des Adaptations Physiologiques aux Activite
´s Physiques, Universite
´de Poitiers, 86034 Poitiers Cedex, France
Received 13 May 2008
Accepted 31 October 2008
Whole-body cryotherapy (WBC) covers a wide range of therapeutic applications and consists of brieﬂy
exposing the body to extremely cold air. In sports medicine, WBC is used to improve recovery from
muscle injury; however, empirical studies on its application to this area are lacking. To ﬁll this gap, we
compared changes in immunological parameters (C3, IgA, IgM, IgG, C-reactive protein, PGE2), cytokines
(IL-2, IL-8, IL-10), adhesion molecules (sICAM-1), and muscle enzymes (creatine kinase [CK], lactate
dehydrogenase [LAD]) before and after WBC in 10 top-level Italian National team rugby players. The
subjects underwent ﬁve sessions on alternate days once daily for 1 week. During the study period, the
training workload was the same as that of the previous weeks. Compared to baseline values,
immunological parameters remained unchanged, while CK and LAD levels signiﬁcantly decreased after
treatment. No alterations in immunological function were observed but there is a decrease in pro-
inﬂammatory cytokine/chemokine and an increase in anti-inﬂammatory cytokine.
As measured by changes in serum CK and LAD concentrations, and cytokines pathway, short-term
cold air exposure was found to improve recovery from exercise-induced muscle injury and/or damage
associated with intense physical training.
&2008 Elsevier Ltd. All rights reserved.
Whole-body cryotherapy (WBC) consists of brief exposure to
extreme cold in a temperature-controlled chamber (110 1C)
(Westerlund et al., 2004). It is applied to relieve pain and
inﬂammatory symptoms caused by numerous disorders, particu-
larly those associated with rheumatic conditions, and is recom-
mended for the treatment of arthritis, ﬁbromyalgia and
ankylosing spondylitis. In sports medicine, WBC has gained wider
acceptance as a method to improve recovery from muscle injury;
however, no controlled studies have been published so far.
WBC has been shown not to be deleterious to lung function
(Smolander et al., 2006) or to decrease antioxidant capacity
´et al., 2005) or propiomelanocortin-related hormones
(Fricke et al., 1988). In a previous study, we demonstrated that
WBC does not enhance hematological values, as measured by
hemoglobin concentration and counts of erythrocytes, reticulo-
cytes, leukocytes, and platelets in peripheral blood (Banﬁ et al.,
2008). Studies investigating the effects of cold exposure on
immune function (Walsh and Whitham, 2006) found that
lymphocyte, monocyte and tumor necrosis factor
increased, whilst concentrations of interleukins IL-6, IL-1
C-reactive protein (CRP) were unchanged after 6 weeks of cold
water immersions (Jansky et al., 1996). Moreover, resting levels of
IL-6, lymphocytes and monocytes were noted to be higher in
subjects accustomed to winter swimming than in inexperienced
¨nen, 2000). Contrary to popular
belief, cold exposure can actually stimulate rather than depress
immune function (Walsh and Whitham, 2006) In general, WBC
does not appear to be harmful; indeed, it may be beneﬁcial for
athletes since prompt recovery from muscle injury is a primary
concern for both athletes and sports physicians alike. Despite the
wealth of literature on rehabilitation techniques, published data
on WBC in rehabilitation programs are scarce. Studying the effects
of WBC can have practical value not only for many physiological
ARTICLE IN PRESS
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Journal of Thermal Biology
0306-4565/$ - see front matter &2008 Elsevier Ltd. All rights reserved.
Corresponding author at: Istituto Ortopedico R. Galeazzi, IRCCS, 20161 Milan,
E-mail address: email@example.com (M.M. Corsi).
Journal of Thermal Biology 34 (2009) 55–59
and clinical purposes, but also for determining clinical signiﬁ-
cance in the context of antidoping testing, since techniques that
accelerate recovery may be classiﬁed as prohibited. Furthermore,
post-WBC treatment changes in biochemical and hematological
parameters could result outside the threshold range imposed by
sports federations and ofﬁcial control agencies, with the athletes
classiﬁed as being doped, or could be interpreted as an attempt to
mask changes caused by illicit treatment different from WBC.
The aim of this study was to determine whether WBC had a
positive effect on musculoskeletal metabolism, recovery from
exertional muscle damage, and immune function. Biochemical
and immunological markers were measured at baseline and after
1 week of WBC treatment (ﬁve once-daily sessions) in 10 male
rugby players selected randomly from the Italian National team.
Ten male athletes (mean age 2672.5 years; mean body-mass
index 27.572.3 kg/m
) underwent once daily WBC treatment for
5 days (Monday–Wednesday, Friday, and Saturday) at the Olympic
Rehabilitation Center of Spa"a (Poland). Wearing minimal cloth-
ing, the subjects were ﬁrst exposed to very cold air (30s at 60 1C)
then to extremely cold air (2 min at 110 1C). They reported an
improved sense of general well-being and no discomfort or
During the study period, the subjects continued with their
regular training. The workload was the same as in the previous
6 weeks. Training consisted of 3 h of daily exercises: 1 h of
maximal training in the morning, followed by 1h of submaximal
effort, then 1 h of submaximal training in the afternoon, in
addition to conditioning exercises.
No illnesses occurred during the study period. Diet was
controlled and identical to that of the previous 6 weeks.
The 10 subjects were chosen randomly from the Italian
National rugby team (30 athletes). All gave informed consent to
the study protocol. Blood samples were drawn by vacutainer
tubes at 8 a.m. on the ﬁrst day of treatment (Monday) and then at
the end of treatment on the following Monday. All subjects
continued with the same training workload as that of the previous
weeks. The time period from the last intense training session was
the same for both blood drawings. The serum samples were
separated within 3 h from drawing and stored at –20 1C until
assayed. All biochemical parameters were measured on a Roche
Modular (Roche, Basel, Switzerland), except for high-sensitivity
CRP (hs-CRP), which was measured nefelometrically on a BN
ProSpec analyzer (Behring, Marburg, Germany). PGE2, cytokines
(IL-2, IL-8, IL-10) and sICAM-1 were measured with ELISA kits
(R&D Systems, Minneapolis, MN, USA) and read on an spectro-
photometer (GDV, Milan, Italy).
Statistical analysis was performed using the paired ttest on a
MedCalc program (Mariawerke, Belgium). Statistical signiﬁcance
was set at po0.05.
Table 1 compares pre- and post-treatment blood chemistry
values. A slight but not signiﬁcant increase in Ig and a slight but
not signiﬁcant decrease in CRP were noted. Lymphocyte and
monocyte counts remained substantially unchanged (p¼n.s.):
44.778.2% versus 37.8710.6% and 9.671.7% versus 9.673.5%,
respectively. LAD and CK both decreased signiﬁcantly:
307.77103.2U/L versus 183.9783.4 U/L and 362.3734.3 U/L
versus 318.4728.6 U/L, respectively. The standard deviation in
the post-treatment values decreased, indicating a greater uni-
formity of data.
The hs-CRP assay is a highly sensitive test for in measuring CRP
levels in inﬂammation associated with rheumatoid arthritis and
cardiac disease. The results of the hs-CRP test were similar to
those obtained with conventional CRP testing. The mean hs-CRP
before and after WBC was 0.8870.40 and 0.7770.40 mg/L,
respectively (p¼n.s.). Table 2 shows pro-inﬂammatory cytokines,
prostacyclin, and adhesion molecule: a slight signiﬁcant decrease
of IL-2 (po0.05) and IL-8 (po0.05), and a very signiﬁcant increase
of IL-10 (po0.05) were noted. PGE2 was signiﬁcantly decreased
(p¼o0.0001). Moreover also sICAM-1 decreased in a very
signiﬁcant way (po0.01) (Fig. 1)
The use of methods other than passive recovery for improving
recovery after intense training and competitions in sports is
growing. In particular, in rugby, the use of cold water, possibly
associated with active recovery (cycling), and the use of cold and
hot water immersion are quite popular. WBC refers to brief
exposure to very cold air for treating symptoms of various
illnesses. In sports medicine, WBC is administered to improve
recovery from muscular trauma. As speciﬁc studies are lacking, we
measured immunological and muscular markers in 10 top-level
rugby players of the Italian National team before and after a
1-week course of daily sessions of WBC. The cold has also been
shown to increased concentrations of anti-inﬂammatory cyto-
kines in peripheral blood: it is suggested to have local and
systemic anti-inﬂammatory effect.
We chose to compare changes in immunoglobulin and CRP
levels because they are reliable indicators of acute or chronic
infection and/or inﬂammation. Widely available in clinical
laboratories, these markers are routinely and easily evaluated in
the general population and in athletes. Lymphocyte and monocyte
counts and plasma IL-6 concentrations are known to be higher in
experienced than in inexperienced winter swimmers, probably
ARTICLE IN PRESS
Serum concentration of immunological markers and muscle enzymes before and
after whole-body cryotherapy (WBC) in 10 top-level rugby players.
Before WBC After WBC Pvalue
IgG (mg/dL) 1262.47196.5 1286.37186.4 n.s.
IgM (mg/dL) 97.9733.7 100.5735.1 n.s.
IgA (mg/dL) 240.17106.2 250.37116.3 n.s.
CRP (mg/dL) 0.7470.43 0.6270.38 n.s.
C3 (U/L) 140.3720.9 142.5721.6 n.s.
CK (U/L) 307.77103.2 183.9783.4 o0.01
LAD (U/L) 362.3734.3 318.4728.6 o0.01
PGE2 (pg/mL) 1162.97292.3 351.57179.9 o0.0001
7values are means7SD. CRP—C-reactive protein; C3—C3 proactivator; CK—
creatine kinase; LAD—lactate dehydrogenase; PGE2—prostaglandin E2.
Serum concentration of cytokines and adhesion molecule before and after whole-
body cryotherapy (WBC) in 10 top-level rugby players.
Before WBC After WBC Pvalue
IL-2 pg/mL 11.3775.91 6.4773.66 o0.05
IL-8 pg/mL 10.3072.31 8.1872.11 o0.05
IL-10pg/mL 41.1573.07 45.8473.55 o0.01
SICAM-1 ng/mL 196.147113.12 74.68744.11 o0.01
IL-2—interleukin-2; IL-8—interleukin-8; IL-10—interleukin-10; sICAM-1—soluble
intercellular adhesion molecule 1.
G. Banﬁ et al. / Journal of Thermal Biology 34 (2009) 55–5956
because of long duration exercise in a cold environment by
experienced subjects. WBC is not characterized by changes in
immunological markers and does not seem to impair immune
function, as measured by using immunological parameters with
paracrine activity (Dugue
¨nen, 2000). It is not clear how
WBC relieves pain and other symptoms of rheumatoid arthritis
and arthropathies in general, but local cryotherapy has been
shown to exert an analgesic effect on and a protective effect
against collagenase on cartilage (Harris and McCroskery, 1974).
The effect of WBC may be linked to alterations of paracrine
molecules rather than to systemic immune functions. In sports
medicine, WBC has gained wider acceptance as a procedure to
improve recovery from muscular trauma; however, controlled
studies on athletes are lacking.
ARTICLE IN PRESS
Before WBC After WBC
Before WBC After WBC
Before WBC After WBC
Before WBC After WBC
Before WBC After WBC
Before WBC After WBC
Before WBC After WBC
400 Before WBC
G. Banﬁ et al. / Journal of Thermal Biology 34 (2009) 55–59 57
Published data suggest that WBC has no detrimental effect on
immunological parameters, although the observation period in
this study was too short to evaluate changes in lymphocyte
involvement and function. Long-term cold water immersion
of healthy males is known to produce slight increases in
, lymphocytes and monocytes (Jansky et al., 1996)
Speciﬁc studies on immunoglobulins during and after WBC are
Several studies have investigated the effect of cold stress on
immune system function. Cold exposure in a climate chamber at
51C induced a small, but signiﬁcant leukocytosis due to an
increase in circulating neutrophils and lymphocytes, accompanied
by natural killer cell activity (Brenner et al., 1999). The authors
remarked that cold exposure had an immunostimulating effect
that was possibly related to an enhanced noradrenaline response
to the cold.
There is, in general, limited evidence indicating that short- or
long-term cold exposure causes immunosuppression. In contrast,
a stimulating effect of cold exposure was found to depend on the
relationship between the decrease in core temperature and the
duration of cold exposure (Walsh and Whitham, 2006) In one
study, IgA and IgM concentrations were reported to be lower
during the ﬁrst 4 months of a 1-year Antarctic expedition
(Gleeson et al., 2000). The change in mucosal immunity was
related to the psychological stress of the expedition, but no
increase in upper respiratory tract infections was observed. It
could be argued that long-term cold exposure, especially when
associated with psychological discomfort and mood modiﬁca-
tions, depresses immunoglobulin production and release, whereas
very short cold exposure does not affect Ig levels. Moreover, cold
inhibits the expression of inﬂammatory mediators; hypothermia
inhibits activation of neutrophils and expression of intercellular
adhesion molecule-1 (ICAM-1), the adhesion and activation of
neutrophils during inﬂammation (Hanusch et al., 2007). Therefore
hypothermia induced expression of the anti-inﬂammatory cyto-
kines IL-10 (Scumpia et al., 2004); hypothermia attenuates the
inﬂammatory response during WBC, thus contributing to its
beneﬁcial role in organ protection (Hofstetter et al., 2007). As
elevated serum CK is a characteristic marker of exertional
rhabdomyolysis, it may be used for measuring the effects of
workload, recovery and possible overtraining. In a previous study
on top-level rugby players, we found that active recovery with leg
immersion in cold water after training produced beneﬁcial effects
and a decrease in serum total CK concentration in comparison
with passive recovery (Banﬁ et al., 2007). These results were in
line with those of Gill et al. (2006) observed in rugby players by
measuring CK in interstitial muscular ﬂuid. It seems that acute
exposure of the whole body to cold air stimulates muscle ﬁber
repair by reducing cell membrane breakdown or increased cell
permeability caused by oxidant agents produced during physical
exercise (Banﬁ et al., 2006). In our subjects, the signiﬁcant
decrease in serum total CK concentration suggested rapid
recovery from muscle damage, since the athletes did not change
the training scheme or workload during the study period.
No published data are available about the behavior of CK and
LAD after WBC. The mechanism underlying the decrease in
muscle enzyme levels is unknown. The normal response of the
human body to cold exposure is accelerated elimination of
triiodothyronine (T3) and activation of the sympathetic nerve
system, which increases the release of noradrenaline in the blood
¨luoto et al., 2005). The augmented T3 catabolism is not
accompanied by activation of the pituitary–thyroid axis during
long-term cold exposure, but rather by a hypothyroid-like status
¨luoto et al., 2005). Concentrations of thyroid-stimulating
hormone, tetraiodothyronine and T3 were unchanged in WBC-
treated patients with rheumatoid arthritis (Zagrobelny et al.,
1992). However, even if short-term cold exposure could inﬂuence
thyroid metabolism, CK is elevated in subclinical hypothyroidism
(Hekimsoy and Oktem, 2005). Thyroid response to cold exposure
could perhaps act through a decreased sensitivity of mitochondria
to ADP and creatine, as well as mitochondrial CK, inﬂuencing the
entire CK (and muscle isoenzyme LAD) metabolism (Athe
´a et al.,
2007). Sustained noradrenaline stimulation during long-term cold
exposure and WBC could play a role in relieving pain and creating
an enhanced sense of well-being. However, in top-level athletes
exercising in a cold environment, there is a simultaneous increase
in noradrenaline and CK levels, with the noradrenaline increase
generally higher than that of CK (seven-fold versus two-fold),
when compared with baseline values (Rønsen et al., 2004)
Noradrenaline stimulation cannot completely explain the CK
decrease in our subjects after WBC treatment, but it may have
triggered a cascade of events that induced the decrease.
WBC had no adverse effect on the immunological status of the
subjects and had a positive effect on CK and LAD levels.
Moreover, there is an increase of anti-inﬂammatory cytokine
IL-10, and a decrese of pro-inﬂammatory cytokine IL-2 and
chemokine IL-8. We conﬁrmed the decrease of sICAM-1 induced
by cold treatment, inducing an anti-inﬂammatory response, also
correlated to decrease of PGE2. PGE2 is synthesized in substantial
amounts at sites of inﬂammation where it acts as a potent
vasodilator and sinergistically with other mediators such as
histamine and bradykinin causes an increase in vascular perme-
ability and edema. Moreover, PGE2 is a central mediator of febrile
response triggered by the inﬂammatory process and intradermal
PGE2 is hyperalgesic in the peripheral nervous system.
The blood chemistry values demonstrate that this treatment
cannot be considered as an illegal or unethical procedure, e.g.,
blood boosting (Banﬁ et al., 2008). Additional studies on
physically active subjects are needed to conﬁrm these ﬁndings,
possibly including case-control protocol.
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