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The Effects of Cold Exposure Training and a Breathing
Exercise on the Inflammatory Response in Humans: A
Pilot Study
Jelle Zwaag, MD, Rick Naaktgeboren, MD, Antonius E. van Herwaarden, PhD,
Peter Pickkers, MD, PhD, and Matthijs Kox, PhD
ABSTRACT
Objective: We previously showed that a training intervention comprising a combination of meditation, exposure to cold, and breathing
exercises enables voluntary activation of the sympathetic nervous system, reflected by profoundly increased plasma epinephrine levels,
and subsequent attenuation of the lipopolysaccharide (LPS)-induced inflammatory response. Several elements of the intervention may
contribute to these effects, namely, two different breathing exercises (either with or without prolonged breath retention) and exposure to
cold. We determined the contribution of these different elements to the observed effects.
Methods: Forty healthy male volunteers were randomized to either a short or an extensive training in both breathing exercises by either the
creator of the training intervention or an independent trainer. The primary outcome was plasma epinephrine levels. In a subsequent study,
48 healthy male volunteers were randomized to cold exposure training, training in the established optimal breathing exercise, a combina-
tion of both, or no training. These 48 participants were subsequently intravenously challenged with 2 ng/kg LPS. The primary outcome
was plasma cytokine levels.
Results: Both breathing exercises were associated with an increase in plasma epinephrine levels, which did not vary as a function of length
of training or the trainer (F(4,152) = 0.53, p=.71,andF(4,152) = 0.92, p= .46, respectively). In the second study, the breathing exercise
also resulted in increased plasmaepinephrine levels. Cold exposure training alone did not relevantly modulate the LPS-induced inflamma-
tory response (F(8,37) = 0.60, p= .77), whereas the breathing exercise led to significantly enhanced anti-inflammatory and attenuated pro-
inflammatory cytokine levels (F(8,37) = 3.80, p= .002). Cold exposure training significantly enhanced the immunomodulatory effects of
the breathing exercise (F(8,37) = 2.57, p=.02).
Conclusions: The combination of cold exposure training and a breathing exercise most potently attenuates the in vivo inflammatory re-
sponse in healthy young males. Our study demonstrates that the immunomodulatory effects of the intervention can be reproduced in a stan-
dardized manner, thereby paving the way for clinical trials.
Tria l R e g istr a t i on: ClinicalTrials.gov identifiers: NCT02417155 and NCT03240497.
Key words: innate immunity, human endotoxemia, cold exposure, breathing exercise.
INTRODUCTION
Previous work from our group revealed that healthy volunteers
who followed a training program were able to voluntarily activate
their sympathetic nervous system and attenuate their inflammatory
response during experimental human endotoxemia, a standardized,
controlled, and reproducible model of systemic inflammation elic-
ited by intravenous administration of bacterial lipopolysaccharide
(LPS) (1). The training program was devised by a Dutch individual,
who holds several world records with regard to withstanding ex-
treme cold, in whom initial indications for the previously described
effects of the intervention were observed (2). The training consists
of three elements, namely, meditation, exposure to cold, and breath-
ing exercises. Trained participants, who practiced the breathing ex-
ercises during experimental endotoxemia, exhibited high plasma
concentrations of epinephrine, which were related to a rapid and
profound increase of the anti-inflammatory cytokine interleukin
Supplemental Digital Content
From the Department of Intensive Care Medicine (Zwaag, Naaktgeboren, Pickkers, Kox), Radboud Center for Infectious Diseases (RCI) (Zwaag,
Pickkers, Kox), and Department of Laboratory Medicine (van Herwaarden), Radboud university medical center, Nijmegen, the Netherlands.
Address correspondence to Matthijs Kox, PhD, Department of Intensive Care Medicine (710), Radboud university medical center, Geert Grooteplein
Zuid 10, PO Box 9101, 6500 HB Nijmegen, the Netherlands. E-mail: matthijs.kox@radboudumc.nl
Received for publication October 28, 2020; revision received November 4, 2021.
DOI: 10.1097/PSY.0000000000001065
Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Psychosomatic Society. This is an open-access
article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permis-
sible to download and share the work provided it is properly cited.The work cannot be changed in any way or used commercially without permission from
the journal.
AUC = area under the curve, BRT = breathing exercise without re-
tention group, CBR = cold exposure and the breathing exercise
without retention group, CEX = cold exposure group, CON = con-
trol group, IL = interleukin, IP = interferon gamma-induced pro-
tein, LPS = lipopolysaccharide, MCP = monocyte chemoattractant
protein, MIP = macrophage inflammatory protein, TNF =tumor
necrosis factor
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Psychosomatic Medicine, V 84 •457-467 457 May 2022
(IL) 10 and subsequent attenuation of the proinflammatory response (e.g.,
plasma levels of tumor necrosis factor [TNF] α, IL-6, and IL-8) (1).
The anti-inflammatory effects of this intervention could repre-
sent a novel treatment modality that may empower patients with
inflammatory conditions, such as autoimmune diseases. However,
there are several questions that need to be addressed first. Most
importantly, it needs to be established which (combination of) el-
ement(s) is/are responsible for the effects observed, as feasibility
may increase if potential users of the intervention would have to
learn and practice less elements, but still attain the same efficacy.
The meditation exercise is likely of limited relevance, as it was a
very minor part of the training program and was not practiced dur-
ing the endotoxemia experiments (1). The breathing exercises both
involved cyclic hyperventilation (1). In one exercise, each cycle of
hyperventilation was followed bybreath retention for up to several
minutes, resulting in profound decreases in oxygen saturation,
whereas in the other exercise, participants only very shortly held
their breath after each cycle of hyperventilation during which all
body muscles were tightened, which was not associated with a de-
crease in oxygen saturation. Because both hyperventilation and
hypoxia have been shown to result in epinephrine release (3–6),
it is unknown which of these exercises is responsible for the ob-
served effects. Furthermore, it is unclear whether it is necessary
to be trained by the creator of the intervention (with regard to the
so-called guru effect, in which the mere presence of an authoritar-
ian figure influences symptomatology (7,8)) and whether or not a
short instruction instead of an extensive training would be suffi-
cient to increase plasma epinephrine levels (1).
In the first part of the current study, we addressed these issues
by investigating the effects of the two different breathing exercises
and different training modalities (i.e., training by the creator of the
intervention versus an independent trainer, and a short instruction
versus extensive training) on plasma epinephrine levels, as these are
implicated to be the main determinant of the anti-inflammatory ef-
fects of the intervention (1). In the second part of this study, we in-
vestigated the effects of the optimal breathing exercise established
in the first part and of cold exposure, both independently and com-
bined, on the inflammatory response during experimental human
endotoxemia. In this highly controlled and reproducible model, a
systemic inflammatory response is elicited by intravenous adminis-
tration of bacterial LPS to healthy volunteers (9). This model is used
to investigate the inflammatory response and possible therapeutics
in sepsis and other inflammatory conditions, but also offers possibil-
ities to study mechanisms underlying cytokine-induced behavioral
changes and to characterize potential targets of therapies against
inflammation-associated depression (10). Cold exposure may in-
fluence the inflammatory response either through a direct,
epinephrine-independent effect or by enhancing epinephrine
levels elicited by the breathing exercise.
By identifying the efficacy of the different training modalities and
elements, this study aims to shed light on the underlying mechanisms
responsible for the previously observed anti-inflammatory effects and
will aid future clinical development of the training intervention.
METHODS
Ethical Approval and Participants
All procedures were approved by the local ethics committee of the Radboud
university medical center (Commissie Mensgebonden Onderzoek—Human
Research Ethics Committee Arnhem-Nijmegen, reference numbers are pro-
vided in the corresponding sections hereinafter) and were conducted in accor-
dance with the Declaration of Helsinki including current revisions and Good
Clinical Practice guidelines. Data were collected between December 2014
and June 2016. Participants were community volunteers, mostly students,
who were recruited through paper leaflets, posters, and online communities
within the campus of the Radboud University in Nijmegen, the Netherlands.
All participants provided written informed consent to participate in the
study and were screened before the start of the experiment to confirm a nor-
mal physical examination, electrocardiography, and routine laboratory
values. Exclusion criteria were prior experience with any of the elements
of the intervention developed by the creator of the intervention or other
breathing, meditation, or cold exposure exercises (including mindfulness,
yoga, exposure to cold showers, and frequent visits to saunafacilities [more
than once per month]). Additional exclusion criteria were use of any med-
ication, smoking, previous spontaneous vagal collapse, use of recreational
drugs within21 days before the experiment day, surgery or trauma with sig-
nificant blood loss or blood donation, hospital admission or surgery with
general anesthesia, participation in another study within 3 months before
the experimental day, or clinically significant acute illness (including infec-
tions) within 4 weeks before the experiment day.
Breathing Exercises Study
Study Design
After ethical approval (reference number: 2014-1374/NL51237.091.14),
40 males provided written informed consent to participate in this prospec-
tive randomized study registered at ClincialTrials.gov (NCT02417155). A
schematic overview of the study is depicted in Figure 1. Participants were
randomized to four different groups (n= 10 per group) by an independent
research nurse using the sealed envelope method: extensive training by the
creator of the intervention, extensive training by an independent trainer,
short training by the creator of the intervention, and short training by an in-
dependent trainer. All participants were trained in both breathing exercises,
with and without the prolonged breath retention (detailed in the Breathing
Exercises section), in the week before the experiment day.
Training Procedures
In the group who received the extensive training by the creator of the inter-
vention, participants were trained every morning for 2 hours for 4 days, and
after these initial 4 days of training, participants were instructed to practice
the learned exercises at home, both analogous to our previous study (1). In
the group who received the short training by the creator of the intervention,
participants were trained for only 2 hours on the morning of the fourth day
(Figure 1), and participants were instructed not to practice the learned exer-
cises at home. The training procedures in the other two groups were exactly
the same, with the exception that the creator was substituted by an indepen-
dent trainer from our research group (R.v.G.), and that participants also re-
ceived a detailed written instruction of both breathing exercises (Appendix,
“Written instructions for breathing exercises,”Supplemental Digital Con-
tent 1, http://links.lww.com/PSYMED/A820).
Breathing Exercises
In the exercise with the prolonged retention of breath (henceforth des-
ignated as “with [+] retention”), participants hyperventilated for an av-
erage of 30 breaths using deep and powerful breaths. Subsequently, the
participants exhaled and held their breath for approximately 2 minutes
(“retention phase”). The duration of breath retention was entirely at the
discretion of the participant. Breath retention was followed by a deep
inhalation breath, which was held for 10 seconds. Subsequently a
new cycle of hyper/hypoventilation began. In the exercise without retention
of breath (henceforth designated as “without [−] retention”), participants also
hyperventilated for an average of 30 times using deep and powerful breaths.
Subsequently, participants held their breath for only 10 seconds, during
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Psychosomatic Medicine, V 84 •457-467 458 May 2022
which all body muscles were tightened, and then a new cycle of hyperventi-
lation was initiated.
Procedures on the Experiment Day
The experiments were conducted at the research unit of the intensive care
department of the Radboud university medical center, and an overview of
the procedures is depicted in Figure 1. To allow for comparison with our
previousstudy (1), participants refrained from caffeine and alcohol 24 hours
before the experiment, and refrained from any intake of food and drinks
10 hours before the experiment. Fasting was maintained throughout the
two breathing exercise sessions. A cannula was placed in the antecubital
vein of the nondominant arm for hydration, and the radial artery of the same
arm was cannulated under local anesthesia (lidocaine HCl 20 mg/mL)
using a 20-gauge arterial catheter for continuous arterial blood pressure
monitoring and blood withdrawal. After a 1-hour rest period, participants
were randomized to start with one of the breathing exercises at 9:00 AM
(morning session): half of the participants started with the exercise with re-
tentions, whereas the other half started with the exercise without retentions.
They performed the exercise for 1.5 hours, after which they rested for
1.5 hours, and the second breathing exercise was started at noon (afternoon
session), which also lasted 1.5 hours. Adherence was assured by a member
of the research team who was present in the room during the entire exper-
iment. Serial blood samples were obtained throughout the experiment
(Figure 1).
Experimental Human Endotoxemia Study
Study Design
After ethical approval (reference number 2016-2312/NL56686.091.16), 48
males provided written informed consent to participate in this prospective
randomized controlled study registered at ClincialTrials.gov (NCT03240497).
A schematic overview of the study is depicted in Figure 2. We used a 2 2
design, in which 48 participants were randomized using the sealed envelope
method to 4 different groups (n= 12 per group): cold exposure (CEX),
breathing exercise without retention (BRT), cold exposure and the breathing
exercise without retention (CBR), and a control group (CON). Participants of
all groups except the control group were trained in the week leading up to the
endotoxemia experiment day.
Training Procedures
An impression of the training procedures is provided (Video, Supplemental
Digital Content 2, http://links.lww.com/PSYMED/A821). All training pro-
cedures were provided by the same independent trainer (R.v.G.). The crea-
tor of the intervention was not involved in the training course. The study
team, including an MD, was present during all training procedures. Participants
in the CEX group followed an intensive 4-day cold exposure training pro-
gram similar to that of our previous study (1), consisting of standing in
snow with bare feet for up to 30 minutes, lying in snow in shorts for up
to 20 minutes, and sitting and swimming in ice-cold water for up to
3 minutes (Video, Supplemental Digital Content 2, http://links.lww.com/
PSYMED/A821). Participants were instructed to end their daily shower
with a period of 60 seconds of cold water. Participants in the BRT group
were trained in the breathing exercise without retentions of breath as de-
scribed previously in the Breathing Exercises section. Similar to the short
training by an independent trainer group in the breathing exercises study
(see the previous section, Training Procedures), the independent trainer
provided an instruction course of 2 hours. Participants were instructed
not to practice the learned exercises at home. Participants randomized to
the CBR group participated in both training procedures, and participants
in the control group did not receive any training.
Procedures on the Endotoxemia Experiment Day
Endotoxemia experiments were conducted at the research unit of the inten-
sive care departmentof the Radboud university medical center according to
our standard protocol (11)also used in our previous study into this interven-
tion (1), and an overview of the procedures is depicted in Figure 2. Partic-
ipants refrained from caffeine, alcohol, and intake of food and drinks in the
same way as the participantsof the breathing exercises study did.A cannula
was placed in the antecubital vein of the nondominant arm for hydration,
and the radial arteryof the same armwas cannulated under local anesthesia
(lidocaine HCl 20 mg/mL; Fresenius Kabi, Zeist, the Netherlands) using a
20-gauge arterial catheter for continuous arterial blood pressure monitoring
and blood withdrawal. Participants received 1.5 L of 2.5% glucose/0.45%
saline solution for 1 hour (prehydration) before LPS administration, followed
by 150 mL/h until the end of the experiment (8 hours after LPS admin-
istration). Participants of the BRT and CBR groups practiced the learned
breathing exercise from 30 minutes before administration of LPS to
FIGURE 1. Schematic overview of the procedures of the breathing exercises study. Dots indicate blood sampling from the arterial catheter
at the corresponding time points.
Immunomodulation by a Training Program
Psychosomatic Medicine, V 84 •457-467 459 May 2022
2.5 hours afterward, identical to our previous study (1). Adherence was as-
sured by a member of the research team that was present in the room during
the entire experiment. Purified LPS (derived from Escherichia coli O:113,
Clinical Center Reference Endotoxin) obtained from the Pharmaceutical
Development Section of the National Institutes of Health (Bethesda,
Maryland) and supplied as a lyophilized powder was reconstituted in
5 mL saline 0.9% for injection and vortex-mixed for 20 minutes before
being administered as an intravenous bolus at a dose of 2 ng/kg body
weight for 1 minute at T= 0 hours at 9:30 AM. Blood samples were se-
rially obtained throughout the experiment (Figure 2).
Epinephrine and Blood Gas Analysis
For circulating epinephrine measurements, blood was collected into lithium hep-
arin tubes and was immediately placed on ice and centrifuged at 2000gfor
10 minutes at 4°C, after which plasma was stored at −80°C until analysis. Plasma
epinephrine concentrations were subsequently measured using high-performance
liquid chromatography with fluorometric detection (12). Blood gas parameters
were analyzed in lithium heparin anticoagulated arterial blood using an i-STAT
Blood Gas Analyzer (Abbot, Hoofddorp, the Netherlands) and CG4+ cartridges.
Plasma Cytokines
EDTA-anticoagulated blood was centrifuged immediately at 2000gfor
10 minutes at 4°C, after which plasma was stored at −80°C until analysis.
Concentrations of TNF-α, IL-6, IL-8, IL-10, interferon gamma–induced
protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), macro-
phage inflammatory protein (MIP) 1α,andMIP-1βwere measured in
one batch using one assay with a simultaneous Luminex assay according
to the manufacturer’s instructions (Milliplex; Merck-Millipore, Billerica,
Massachusetts) on a Magpix instrument (Luminex Corporation, Austin,
Texas). Intra-assay % coefficient of variation of the measured cytokines
as provided by the manufacturer range from 1.5 to 2.6, whereas interassay
% coefficients of variation ranged from 3.5 to 18.3. The detection range
was 3.2 to 10,000 pg/mL for cytokines TNF-α,IL-10,IP-10,MCP1,
MIP1a, and MIP1b and 1.4 to 10,000 pg/mL for IL-6 and IL-8. Samples
below the detection limit were imputed by 3.2 and 1.4 pg/mL, respectively;
no samples were above the upper detection limit.
Hemodynamic Parameters, Symptom Score, and
Temperature
Heart rate (three-lead electrocardiogram), blood pressure (intra-arterial can-
nula), respiratory rate, and oxygen saturation (pulse oximetry) data were
recorded from a Philips MP50 patient monitor (Eindhoven, the Netherlands)
every 30 seconds by a custom in-house–developed data recording system.
LPS-induced flu-like symptoms (headache, nausea, shivering, muscle, and
back pain) were scored every 30 minutes on a 6-point Likert scale (0 = no
symptoms, 5 = worst ever experienced, in case of vomiting 3 points were
added), forming an arbitrary total symptom score with a maximum of 28
points. Body temperature was determined every 30 minutes using an infra-
red tympanic thermometer (First-Temp Genius; Sherwood Medical, Nor-
folk, Nebraska).
Calculations and Statistical Analysis
Data are expressed as median and interquartile range or mean and 95%
confidence interval, based on their distribution calculated by Shapiro-
Wilk tests. For the sample size of the endotoxemia study, we wished
to remain in line with our previous published endotoxemia study on this
intervention (1), in which 12 participants per group were included. We
calculated the achieved power using previous endotoxemia data of our
group on the archetypal pr oinflammatory cytokin e TNF-α.Themeanof
the TNF-αresponse (area under the time-concentration curve [AUC]) was
970 arbitrary units with a standard deviation of 300 arbitrary units (31% of
the mean). Using these values, a detectable contrast (effect size) of 40%
(388 arbitrary units), a two-sided αof .05, and 12 participants per group
in an unpaired ttest design, a power of 86%is achieved. There were no out-
liers that needed to be removed from any analysis. In the breathing exer-
cises study, of the total of 280 sample moments, there were three missing
values in the blood gas parameters because of technical issues. In the
endotoxemia study, there was one missing value in the blood gas pa-
rameters (of a total of 312 sample moments) and five missing values
in the epinephrine data (of a total of 432 sample moments), all because
of technical issues. Serial data were analyzed using linear mixed-
models analysis ( pvalues of “time by column factors”are depicted in
the figures, whereas theresults of post hoc Sidak multiple comparison tests
[only performed in case time by column, the pvalue was <.05] to evaluate
differences at individual time points are provided in the supplemental ta-
bles, (Supplemental Digital Content 1, http://links.lww.com/PSYMED/
A820). AUCs were calculated on a per-participant basis using the “Area
under Curve”function in GraphPad Prism 8.0 (GraphPad Software,
San Diego, California) to provide an integral measure of the cytokine
responses. Multivariate multiple linear regression, entering AUC cytokine
responses as dependent variables and the different groups as independent
variables, was performed to assess the effects of cold exposure training
and the breathing exercises on plasma levels of all measured cytokines.
FIGURE 2. Schematic overview of the procedures of the human endotoxemia study. Dots indicate blood sampling from the arterial
catheter at the corresponding time points. LPS = lipopolysaccharide.
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Psychosomatic Medicine, V 84 •457-467 460 May 2022
Demographic characteristics were analyzed using Kruskal-Wallis tests.
Apvalue <.05 was considered statistic ally significant. Calculations an d
statistical analysis were performed using GraphPad Prism version 8.3.0
and SPSS v25.0.0.1 (IBM Corp, Armonk, New York).
RESULTS
Breathing Exercises Study
Demographic Characteristics
Demographic characteristics of the participants are listed in Table 1
and were not different between the study groups.
Plasma Epinephrine Levels and Blood Gas Parameters
Changes in blood gas parameters were identical during both the
morning and afternoon sessions across all groups (saturation:
F(6,462) = 0.74, p= .62; oxygen partial pressure [pO
2
]: F
(6,463) = 0.98, p=.44;pH:F(6,463) = 0.56, p= .76; and car-
bon dioxide partial pressure [pCO
2
]: F(6,463) = 1.26, p=.28;
Figures S1A–D and Table S5, Supplemental Digital Content 1,
http://links.lww.com/PSYMED/A820). During the morning session,
plasma epinephrine levels sharply increased upon initiation of the
breathing exercises across all groups (from 0.51 [0.33–0.72] nmol/L
at T=0to1.01[0.64–1.48] nmol/L at T=0.5,p< .0001; Figure
S1E and Table S5, http://links.lww.com/PSYMED/A820). We
previously hypothesized that this initial increase in epinephrine levels
is the main driving factor inducing the anti-inflammatory phenotype
(see Figure 5 in Ref. (1)). Epinephrine levels remained elevated for
as long as the participants practiced the exercises in the morning
(0.87 [0.51–1.24] nmol/L and 0.99 [0.56–1.68] nmol/L at T=1,
and 1.5 hours, respectively; Figure S1E and Table S5, http://links.
lww.com/PSYMED/A820). In contrast, during the afternoon session
plasma epinephrine levels failed to rapidly increase after commencing
the breathing exercises, although concentrations were slightly ele-
vated at later time points (T=0:0.48[0.33–0.65] nmol/L, T= 0.5:
0.44 [0.30–0.73] nmol/L, T= 1: 0.54 [0.38–1.12] nmol/L and
T= 1.5: 0.75 [0.54–1.26] nmol/L; Figure S1E and Table S5,
http://links.lww.com/PSYMED/A820). Statistical comparison of
plasma epinephrine levels over time between the morning and after-
noon session yielded a highly significant difference (F(4,312) = 6.42,
p< .001; Figure S1E and Table S5, http://links.lww.com/PSYMED/
A820). Because o f these find ing s, we res tricted all further analy ses
to data obtained during the morning session.
A comparison of the breathing exercises with and without re-
tention revealed that only the exercise with retention resulted in
profound decreases in oxygen saturation levels at the end of each
TABL E 1 . Demographic Characteristics
Breathing Exercises Study
All Participants
(n=40)
Short Training
by Independent
Trainer (n=10)
Extensive Training
by Independent
Trainer (n=10)
Short Training by Creator
(n=10)
Extensive Training
by Creator (n=10) p
Age, y 21
[19–24]
20
[19–22]
22
[19–26]
21
[20–23]
23
[19–26]
.46
BMI, kg/m
2
22.9
[21.4–24.2]
22.5
[21.5–24.8]
23.9
[21.1–25.0]
23.8
[22.2–24.6]
22.3
[20.1–23.6]
.60
Systolic blood pressure,
mm Hg
140
[128–145]
135
[123–148]
143
[128–147]
146
[137–150]
135
[131–140]
.26
Diastolic blood pressure,
mm Hg
71
[64–79]
71
[61–77]
77
[70–85]
69
[62–81]
69
[62–81]
.22
Heart rate, bpm 77
[60–88]
77
[69–84]
86
[59–103]
77
[53–89]
63
[54–82]
.27
Endotoxemia Study
All Participants
(n=48)
CON
(n=12)
Cold Exposure (CEX)
(n=12)
Breathing Exercise (BRT)
(n=12)
Cold Exposure and
Breathing Exercise (CBR)
(n=12) p
Age, y 22
[20–24]
22
[20–22]
23
[20–26]
22
[20–24]
23
[20–25]
.86
BMI, kg/m
2
23.3
[22.2–24.6]
23.1
[22.2–23.9]
22.8
[21.0–24.2]
23.5
[22.7–24.7]
24.5
[22.5–25.6]
.37
Systolic blood pressure,
mm Hg
140
[136–152]
137
[122–156]
142
[137–155]
140
[136–152]
144
[136–152]
.88
Diastolic blood pressure,
mm Hg
72
[64–80]
73
[66–82]
72
[64–81]
70
[62–75]
77
[68–82]
.35
Heart rate, bpm 64
[56–71]
66
[59–75]
65
[56–73]
62
[56–66]
62
[51–67]
.33
Data were obtained using the screening visit and are presented as median [interquartile range].
pValues were calculated using Kruskal-Wallis tests.
BMI = body massindex; bpm = beats per minute; CON = control group; CEX = coldexposure group; BRT = breathing exercisegroup; CBR: cold exposure and breathing exercise
group.
Immunomodulation by a Training Program
Psychosomatic Medicine, V 84 •457-467 461 May 2022
retention phase (from 98% ± 0.2% at T= 0 to 67% ± 5%, 58 ± 3%
and73±4%,atT= 0.5, 1, and 1.5 hours, respectively; F
(6,224) = 31.50, p< .001; Figure 3A and Table S1, http://links.lww.
com/PSYMED/A820). Accor dingly, sharp decreases in pO
2
were
observed in this group (F(6,225) = 18.43, p< .001; Figure 3B
and Table S1, http://links.lww.com/PSYMED/A820). pH and
pCO
2
were largely comparable between the two breathing exer-
cises, with only a slight but statistically significant difference at
the last measured time point (pH: F(6,225) = 7.96, p<.001;
pCO
2
:F(6,225) = 4.67, p< .001; Figures 3C, D, and Table S1,
http://links.lww.com/PSYMED/A820). The initial increase in
plasma epinephrine levels was comparable between both breath-
ing exercises (from 0.51 [0.38–0.75] nmol/L at T=0to0.98
[0.67–1.78] nmol/L at T= 0.5 in the participants performing the
breathing exercise with breath retention, and from 0.51 [0.32–
0.68] nmol/L at T= 0 to 1.01 [0.63–1.46] nmol/L at T= 0.5 in
the participants performing the breathing exercise without reten-
tion, p> .99; Figure 3E and Table S1, http://links.lww.com/
PSYMED/A820). However, the increase in plasma epinephrine
concentrations was slightly more sustained in the participants
practicing the breathing exercise with retention, resulting in signif-
icantly higher levels at T= 1.5 compared with participants practicing
the exercise without retention (F(4,152) = 4.19, p= .003; Figure 3E
and Table S1, http://links.lww.com/PSYMED/A820).
Blood gas parameters and plasma epinephrine levels were not
statistically different between the participants trained by an inde-
pendent trainer compared with participants trained by the creator
of the intervention (saturation: F(6,224) = 0.55, p=.76;p
O
2
:
F(6,225) = 0.26, p=.90;pH:F(6,225) = 0.59, p= .86; pCO
2
:
F(6,225) = 1.57, p= .24; epinephrine: F(4,152) = 0.92, p=.46;
Figure S2, http://links.lww.com/PSYMED/A820). In addition,
no significant differences in these parameters were found between
the participants who received the short training versus the long train-
ing (saturation: F(6,224) = 0.28, p=.95;p
O
2
:F(6,225) = 0.59,
p=.74;pH:F(6,225) = 1.30, p=.26;p
CO
2
:F(6,210) = 0.83,
p= .55; epinephrine: F(4,152) = 0.53, p= .71; Figure S3, http://
links.lww.com/PSYMED/A820).
Based on these results, we conclude that the magnitude of the
initial increase in epinephrine levels, which we previously showed
to be a main determinant of the anti-inflammatory phenotype (1),
is not dependent on prolonged breath retention. Furthermore, neither
training by the creator of the intervention nor a long training program
is required to attain the pronounced epinephrine response. Hence, we
used the training modality consisting of a short training by an inde-
pendent trainer in only the breathing exercise without breath retention
for the subsequent experimental human endotoxemia study.
Experimental Human Endotoxemia Study
Demographic Characteristics
Demographic characteristics of the participants are listed in Table 1
and were not different between groups.
Blood Gas Parameters and Plasma Epinephrine Levels
pCO
2
levels slightly decreased over time in the groups that did not
practice the breathing exercise (CEX and CON groups; Figures 4A–C),
which may reflect a small increase in breathing frequency after LPS
administration. However, no significant changes in this or any of the
other arterial blood gas parameters were observed over time in these
groups, and all values remained within the reference ranges (CEX
and CON groups; Figures 4A–C). In contrast, blood gas parameters
were profoundly altered in the BRTand CBR groups upon initiation
of the breathing exercise and normalized quickly after cessation. pH
increased from 7.46 ± 0.04 (BRT) and 7.40 ± 0.004 (CBR) at
FIGURE 3. Arterial blood gas parameters and plasma epinephrine levels during the breathing exercises study: influence of breathing
exercise. A, Oxygen saturation. B, Oxygen partial pressure (pO
2
). C, pH. D, Carbon dioxide partial pressure (pCO
2
). −retention: data obtained
during the first breathing exercise on the experiment day (breathing exercise 1, Figure 1) from participants performing the breathing exercise
without prolonged retention of breath. + retention: data obtained during the first breathing exercise on the experiment day (breathing exercise 1,
Figure 1) from participants performing the breathing exercise with prolonged retention of breath. Data are presented as mean ± 95% confidence
interval (panels A–D) or median and interquartile range (panel E) of 20 participants per group, and pvalues depicted in the graphs
represent the between-group comparison calculated using linear mixed-models analysis (time by column factor). Epinephrine data were
log-transformed before analysis. For comparisons that yielded time by column factor pvalues <.05, results of post hoc analyses
performed using the Sidak multiple comparison test are reported in Table S1, http://links.lww.com/PSYMED/A820.
ORIGINAL ARTICLE
Psychosomatic Medicine, V 84 •457-467 462 May 2022
baseline to 7.72 ± 0.02 (BRT) and 7.70 ± 0.01 (CBR) 15 minutes
after the start of the breathing exercise, resulting in significant
higher pH during the execution of the breathing exercises in both
groups compared with the CON group (BRT: F(3,88) = 27.80,
p< .01; CBR: F(3,66) = 82.13, p< .01; Figure 4A and Table
S2, http://links.lww.com/PSYMED/A820). Oxygen saturation in-
creased from 98% (98%–99%; BRT) and 99% (98%–100%;
CBR) at baseline to 100% (100%–100%; BRT) and 100%
(100%–100%; CBR) 15 minutes into practicing of the breathing
exercise (BRT: F(3,88) = 12.67, p<.001versusCON;CBR:F
(3,88) = 6.49), p< .001 versus CON; Figure 4B and Table S2,
http://links.lww.com/PSYMED/A820). pCO
2
dropped from
5.16 ± 0.1 kPa (BRT) and 5.17 ± 0.01 kPa (CBR) at baseline to
1.74 ± 0.06 kPa (BRT) and 1.99 ± 0.07 kPa (CBR) 15 minutes
after the start of the breathing exercise (BRT: F(3,66) = 95.23,
p< .001 versus CON; CBR: F(3,66) = 91.04, p<.001versusCON;
Figure 4C and Table S2, http://links.lww.com/PSYMED/A820).
Baseline plasma epinephrine levels were comparable between
all four groups (all pvalues >.05; Figure 4D and Table S2,
http://links.lww.com/PSYMED/A820). Concentrations increased
during human endotoxemia in all groups, with peak values ob-
served 1.5 hours after administration of LPS (Figure 4D and Table
S2, http://links.lww.com/PSYMED/A820). There were no differences
in plasma epinephrine levels over time between the CEX and
CON groups (F(8,160) = 0.94, p= .48). However, in both groups
of participants who practiced the breathing exercises, the increase
in plasma epinephrine commenced much earlier and was signifi-
cantly more pronounced than in the CEX and CON groups that
did not exercise the breathing exercise (BRT F(8,160) = 2.11,
p= .04 versus CON; CBR: F(8,160) = 3.51 p= .01 versus CON;
Figure 4D and Table S2, http://links.lww.com/PSYMED/A820).
Hemodynamic Parameters, Temperature, and
Symptoms
Experimental endotoxemia resulted in a gradual increase in heart
rate in the CEX and CON groups, with no differences between these
two groups (F(18,396) = 0.61, p= .89, Figure 5A). In the two
groups that performed the breathing exercise (BRT and CBR
groups), a sharp increase in heart rate was observed immediately
after the start of the first hyperventilation cycle, and this effect en-
sued during most of the period that the participants practiced the
exercise, resulting in a significant higher heart rate during the ex-
periment compared with the CON group (BRT: F(18,396) = 4.78,
p< .001; CBR: F(18,396) = 3.97, p< .001; Figure 5A and Table
S3, http://links.lww.com/PSYMED/A820). After cessation of the
breathing exercises, the heart rate data of the BRT and CBR groups
were similar to that of the CEX and CON groups. Expectedly, mean
arterial pressuregradually decreased in all groups (Figure 5B and
Table S3, http://links.lww.com/PSYMED/A820), and no clear
differences between any of the groups were present. Although
FIGURE 4. Arterial blood gas parameters and plasma epinephrine levels during human endotoxemia. A, pH. B, Oxygen saturation. C,
pCO
2
. D, Plasma epinephrine concentrations. The gray box indicates the period during which the trained participants practiced the
breathing exercise (BRT and CBR groups only). Data are presented as mean ± 95% confidence interval (panels A–C) or median and
interquartile range of 12 participants per groups. pValues depicted next to the legend represent the comparison of that group with the
control group over time, calculated using linear mixed-models analysis on log-transformed data (time by column factor). Epinephrine
data were log-transformed before analysis. Significant pvalues are shown in bold. For comparisons that yielded time by column factor
pvalues <.05, results of post hoc analyses performed using the Sidak multiple comparison test are reported in Table S2, http://links.
lww.com/PSYMED/A820. BRT = breathing exercise group; CBR = cold exposure and breathing exercise group; CEX = cold exposure
group; CON = control group; pCO
2
= carbon dioxide partial pressure.
Immunomodulation by a Training Program
Psychosomatic Medicine, V 84 •457-467 463 May 2022
there was a statistically significant difference between the BRT
and CON groups in mean arterial pressure over time (F
(18,396) = 1.93, p= .01, Figure 5B), post hoc analysis did not re-
veal significance at any of the individual time points (Table S3,
http://links.lww.com/PSYMED/A820). An LPS-induced mean in-
crease in tympanic temperature of 1.8°C ± 0.1°C was observed
across all groups (Figure 5C). Although peak temperatures were
similar between the CON (38.8°C ± 0.1°C) and the three inter-
vention groups (BRT: 38.8°C ± 0.2°C, p=.94;CEX:38.6°
C±0.2°C,p= .44; CBR: 38.7°C ± 0.2°C, p= .73), these were
attained significantly earlier in the BRT group (F
(18,396) = 1.96, p= .01 versus CON; Figure 5C and Table
S3, http://links.lww.com/PSYMED/A820). Administration of
LPS resulted in flu-like symptoms in all groups (Figure 5D). Peak
symptom scores were comparable between the CON (9.3 ± 1.3),
BRT (9.4 ± 1.5, p= .70), and CBR (7.04 ± 1.2, p= .21) groups,
but significantly lower in the CEX group (5.5 ± 0.8, p=.017).
Symptoms resolved significantly more rapidly in all three intervention
groups compared with the CON group (BRT: F(18,396) = 4.47, p< .001;
CEX: F(18,396) = 1.96, p= .01; CBR: F(18,396) = 2.29, p= .002;
Figure 5D and Table S3, http://links.lww.com/PSYMED/A820).
Plasma Cytokines
Because of the absence of an inflammatory response before LPS
administration and waning of this response multiple hours after
the LPS challenge, 349 of a total of 4224 cytokine measurements
(8%) fell below the lower limit of detection. As expected, plasma
concentrations of the anti-inflammatory cytokine IL-10 and the
proinflammatory cytokines TNF-α,IL-6,IL-8,IP-10,MCP-1,
MIP-1α, and MIP-1βincreased after LPS administration in
all groups (Figure 6 and Table S4, Figure S4 and Table S6,
http://links.lww.com/PSYMED/A820). In the CBR group, IL-10
levels were significantly higher compared with the CON group
(mean increase in AUC of +44%, F(10,220) = 2.20, p=.02;
Figure 6D and Table S4, http://links.lww.com/PSYMED/A820).
Furthermore, concentrations of proinflammatory cytokines in this
group were significantly attenuated compared with the CON group
(mean decrease in AUC of TNF-α:−32%, F(10,220) = 2.09,
p=.03;IL-6:−35%, F(10,220) = 2.06, p=.03;IL-8:−30%, F
(10,220) = 3.97, p< .001; IP-10: −48%, F(10,220) = 7.99,
p<.001;MCP-1:−29%, F(10,220) = 4.64, p<.001;MIP-1α:
−35%, F(10,220) = 6.25, p<.001;MIP-1β:−30%, F(10,220),
p< .001; Figures 6B–D and Table S4, and Figure S4 and Table
S6, http://links.lww.com/PSYMED/A820). When comparing the
BRT group with the control group, similar but less pronounced effects
on plasma cytokines were observed, reaching statistical significance
compared with the CON group for proinflammatory cytokines
IL-6 (−34%, F(10,220) = 1.95, p= .04), IL-8 (−14%, F
(10,220) = 2.25, p= .02), IP-10 (−48%, F(10,220) = 9.42,
p< .001), MCP-1 (−37%, F(10,220) = 6.07, p< .001), MIP-1α
FIGURE 5. Cardiorespiratory parameters, tympanic temperature, and symptoms during human endotoxemia. A, Heart rate. B, MAP. C,
Tympanic temperature. D, Score of self-reported symptoms. The gray box indicates the period during which the trained participants
practiced the breathing exercise (BRT and CBR groups only). Data are expressed as mean ± 95% confidence interval of 12 participants
per group. pValues depicted next to the legend represent the comparison of that group with the control group over time, calculated
using linear mixed-models analysis (time by column factor). Significant pvalues are shown in bold. For comparisons that yielded time
by column factor pvalues <.05, results of post hoc analyses performed using Sidak multiple comparison test are reported in Table S3,
http://links.lww.com/PSYMED/A820. CON = control group; BRT = breathing exercise group; CBR = cold exposure and breathing
exercise group; CEX = cold exposure group; MAP = mean arterial pressure.
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Psychosomatic Medicine, V 84 •457-467 464 May 2022
(−37%, F(10,220) = 6.59, p< .001, and MIP-1β(−28%, F
(10,220) = 2.24, p= .02), but not for IL-10 (+17%, F
(10,220) = 1.42, p= .17). In the CEX group, only levels of
MCP-1 were significantly lower than in the CON group (−25%,
F(10,220) = 4.75, p<.001).
In accordance with the previously described results, a multivar-
iate analysis yielded a significant effect of the breathing exercise, as
well as the interaction between cold exposure training and breathing
exercise on the integral cytokine response (breathing exercise:
F(8,37) = 3.804, p=.002;Wilk’sΛ= 0.549, partial η
2
= 0.451;
cold exposure by breathing exercise: F(8,37) = 2.571, p=.024;
Wilk’sΛ= 0.649, partial η
2
= 0.357). Cold exposure alone did not
significantly affect the integral cytokine response: F(8,37) = 0.603,
p= .769, Wilk’sΛ= 0.885, partial η
2
= 0.115.
DISCUSSION
In this study, we investigated the effects of different aspects of a
training program, which was previously shown toallow for volun-
tary activation of the sympathetic nervous system and attenuation
of the inflammatory response. First, we showed that, although ar-
terial blood saturation levels and pO
2
were significantly lower
when participants performed the breathing exercise with prolonged
breath retention compared with that without, plasma epinephrine
levels increased with a similar magnitude shortly after initiation of
both breathing exercises. Second, we demonstrated that the previously
observed physiological and immunological effects (1) are independent
from either the length of training or the individual who provides
it. Third, our data signify that the combination of the breathing ex-
ercise and cold exposure training is most effective in attenuating
the inflammatory response during human endotoxemia.
As the magnitude of the initial increase in plasma epinephrine
concentrations was similar for the breathing exercises with and
without prolonged breath retention. The cyclic hypoxia caused
by the exercise with prolonged breath retention is therefore un-
likely to be an important factor in the observed epinephrine re-
sponse. In accordance, hyperventilation itself and the subsequent
shift in acid-base balance have been shown to increase plasma cat-
echolamines in the absence of hypoxia, and an important role for
bicarbonate has been implicated (6,13). Nevertheless, because cat-
echolamine release from the adrenal chromaffin cells is dependent
on a combination of neural, hormonal, redox, and immune signal-
ing pathways (14,15), the exact mechanism behind the epineph-
rine release induced by the breathing exercise remains elusive.
The finding that neither the duration of the training nor the trainer
who provides it affected any of the measured parameters signifies
that the breathing exercise is easy to learn within a time frame of 2
hours. These findings may greatly facilitate uncomplicated imple-
mentation of the training program in clinical studies.
Our data clearly demonstrate that the breathing exerciseplays a
pivotal role in the anti-inflammatory effect of the training intervention.
FIGURE 6. Plasma concentrations of inflammatory cytokines during human endotoxemia. A, TNF-α.B,IL-6.C,IL-8.D,IL-10.The
gray box indicates the period during which the trained participants practiced the breathing exercise (BRT and CBR groups only). Data
are presented as mean ± 95% confidence interval of 12 participants per group. pValues depicted next to the legend represent the
comparison of that group with the control group over time, calculated using linear mixed-models analysis (time by column factor).
Significant pvalues are shown in bold. For comparisons that yielded time by column factor pvalues <.05, results of post hoc analyses
performed using the Sidak multiple comparison test are reported in Table S4, http://links.lww.com/PSYMED/A820. BRT = breathing
exercise group; CBR: cold exposure and breathing exercise group; CEX = cold exposure group; CON = control group; IL = interleukin;
TNF = tumor necrosis factor.
Immunomodulation by a Training Program
Psychosomatic Medicine, V 84 •457-467 465 May 2022
Nevertheless, although cold exposure training alone had minimal
effects on the cytokine response, it significantly potentiated the breath-
ing exercise-induced anti-inflammatory effects. Because plasma epi-
nephrine levels in our study were comparable between the groups
practicing the breathing exercises with or without prior cold exposure
training, other mechanisms are likely involved. Noteworthy, despite
little effects on the cytokine response, participants in the cold exposure
training group reported remarkably less symptoms compared with the
control group as well as to the other two groups. In accordance, other
studies reporting symptoms during repeated exposures to cold found
similar attenuation of symptoms such as discomfort and shivering
(16,17). This is possibly part of a stress-induced analgesic response
to cold (18) Symptoms, especially headache, were more pronounced
during practicing of the breathing exercise, likely resulting from the
hyperventilation-induced changes in pCO
2
and pH. After cessa-
tion of the breathing exercise, a sharp decrease of symptoms was
observed and flu-like symptoms resolved more rapidly compared
with the control group.
In the endotoxemia study, the increase in plasma epinephrine
concentrations observed after initiation of the breathing exercises
described in the present work was similar in magnitude to that in
our previous endotoxemia study (1). Nevertheless, epinephrine
levels before the start of the breathing exercises were higher in
the past work (1). Effects on the cytokine response in the com-
bined cold exposure and breathing group in the current study were
largely comparable to our previous work, in which participants
were also trained in both exercises (1), although the magnitude
of the immunomodulatory effects was less pronounced, with the
anti-inflammatory IL-10 response augmented by 44% instead of
194% in Ref. (1) and proinflammatory cytokines attenuated by ap-
proximately 30% as opposed to more than 50% in Ref. (1). There
are several possible explanations for this discrepancy. First, the
previously mentioned higher baseline plasma epinephrine concen-
trations could play a role (1), which may in turn have triggered a
more pronounced early IL-10 release and subsequent stronger at-
tenuation of the proinflammatory response. Second, our data from
the breathing exercises study show that, although the initial in-
crease in epinephrine levels was similar in response to the exercise
with and without breath retention, it was more prolonged in the
former. Despite that fact that we previously showed that the initial
epinephrine increase is a main determinant of the anti-inflammatory
phenotype (1), we cannot exclude the possibility that a more pro-
longed increase has a more pronounced effect. Third, the hypoxia
induced by breath retention in our previous study may have directly
(i.e., independently from epinephrine) modulated the inflammatory
response, as our group has recently demonstrated that hypoxia en-
hances IL-10 release and attenuates the proinflammatory response
via enhanced adenosine release (19). In this light, future studies into
the training intervention should still consider including the exercise
with prolonged breath retention. This exercise may nevertheless be
less preferable from a safety perspective, as the profound cyclic de-
creases in oxygen saturation may present risks for patients with for
instance cardiovascular conditions.
A striking finding from the breathing exercises study was that
the profound increase in plasma epinephrine levels only occurred
during the first session in the morning, not during the second ses-
sion performed in the afternoon after a 1.5-hour resting period.
Nevertheless, the saturation, pO
2
,pCO
2
, and pH were identical be-
tween the morning and the afternoon sessions. Therefore, the lack
of a profound increase of plasma epinephrine levels during the af-
ternoon session may be due to adaptation of the stress response, re-
sulting in lower epinephrine release by the adrenal gland in re-
sponse to repeated application of the same stressor, a phenomenon
that has been described in animals (20). Alternatively, because the
synthesis and storage of catecholamines mainly take place within
chromaffin cells of the adrenal medulla, it may be speculated that
the breathing exercises deplete the intravesicular stores in the cyto-
plasm of these cells (14,15). Although animal experiments have
shown that fully depleted catecholamine stores can be replenished
within 2 hours (21), this may take longer in humans. In any case, if
stores are indeed depleted by the breathing exercise, replenishment
must occur within a relatively short time frame (<24 hours), as par-
ticipants of this study, as well as our previous study (1) practiced the
breathing exercise daily in the week leading up to the experiment in
which the plasma epinephrine concentrations were measured.
Several limitations of our work need to be addressed. First, we
studied groups of healthy young male adults, not (older) patients
with possible comorbidities, who represent the intended target
group for this intervention. We only included male participants be-
cause there are considerable differences in the cytokine response
to LPS between the sexes (22). This could be due to menstrual
cycle–related hormonal variations that affect immunity. Because
human endotoxemia studies are very labor-intensive and expen-
sive, and for ethical reasons (to expose as few volunteers as possi-
ble to endotoxemia), nearly all of our LPS studies are restricted to
males to increase homogeneity and reduce sample size. Of note,
there are no data available regarding the influence of sex differ-
ences on training-induced modulation of the immune response.
Furthermore, the creator of the intervention has trained both men
and women, with no apparent differences in competence regarding
completion of the training exercises (observational data). Although
upcoming studies into this intervention should also include females,
this study provides essential information in terms of designing the
most safe and optimal training protocol for use in these future inves-
tigations. Second, the autoimmune response observed in patients
with chronic inflammatory conditions clearly differs from that elic-
ited by LPS administration, which models an acute inflammatory re-
sponse to a bacterial infection. However, several drugs currently
used in patients with inflammatory conditions such as rheumatoid
arthritis, ankylosing spondylitis, and psoriatic arthritis are aimed at
reducing the release of several proinflammatory cytokines (23), on
which the studied intervention has a substantial suppressive effect.
Furthermore, in vivo human efficacy of many biologics used in
the treatment of auto-inflammatory disorders, such as anakinra
and infliximab, was first established in the experimental human
endotoxemia model (24,25), illustrating that it has value for these
diseases. Third, in the breathing exercises study, we did not include
a control group that was not trained. We chose not to because the
breathing exercises study was designed to primarily investigate
what specific exercise caused the increase in epinephrine during
the combination of breathing exercises performed by the partici-
pants in our original study (1) and whether training by the creator
of the intervention is required. Therefore, we compared different
breathing exercises and training modalities head-to-head. Further-
more, the participants acted as their own controls by measuring
epinephrine levels before the start of the breathing exercise on
the experiment day. Fourth, although psychological, social, or be-
havioral factors may also be influenced by this intervention, the
ORIGINAL ARTICLE
Psychosomatic Medicine, V 84 •457-467 466 May 2022
focus of the present study was on physical outcomes. Because the
intervention is ultimately aimed at alleviating symptoms among
individuals with chronic disease, future studies should also evalu-
ate whether the beneficial physiological effects of the intervention
are offset, for example, by anxiety. Fifth, the reliability of the cy-
tokine assay could not be confirmed because samples were not
run in duplicate. Finally, the control group in the current study
did not undergo any form of training. It must be acknowledged
that a training program or other intervention guaranteed not to in-
fluence the sympathetic nervous system and immuneresponse, but
that does result in matching expectations compared with the inter-
vention groups, would represent a more optimal control condition.
Possibilities may entail mindfulness training, self-affirmation, ed-
ucational materials, and placebo injection of a purported beneficial
substance. Furthermore, in future studies, a dismantling design
could be considered in which the component conditions are com-
pared with the full protocol. Another point related to the control
conditions is that we did not record the frequency of spontaneous
(deep) breaths in the endotoxemia study. Because endotoxemia
has been shown to increase deep breath frequency (26), this may
have introduced bias in the control and cold exposure training
groups. However, our pCO
2
data indicate that the LPS-induced in-
crease in (deep) breathing frequency was limited at most. Further-
more, if anything, it would have led to an underestimation of the
effect of the learned breathing exercises and therefore does not
compromise the validity of our findings.
In conclusion, the present study corroborates previous findings
that voluntary activation of the sympathetic nervous system, atten-
uation of the proinflammatory response, and alleviation of symp-
toms during experimental human endotoxemia are possible after
following a training program consisting of cold exposure and a
breathing exercise. Furthermore, these interventions can be pro-
vided by an independent trainer and acquired within a short time
frame. Although these results provide an important next step in
the clinical development of this intervention, they will need to
be replicated and generalized before this intervention can be con-
sidered appropriate for application in clinical populations with
chronic disease.
We thank the independent trainer Rogier van Groenendael for
his contribution to this study and Remi Beunders forediting of the
video illustrating the training procedures.
Source of Funding and Conflicts of Interest: The authors report
no conflicts of interest. Thisstudy was internally funded by the De-
partment of Intensive Care Medicine, Radboud university medical
center, Nijmegen, the Netherlands.
Author contributions: study design/planning: J.Z., A.E.v.H.,
P.P., M.K.; study conduct: J.Z., R.N., P.P., M.K.; data analysis: J.Z.,
M.K.; writing the paper: J.Z., M.K.; critical revision: A.E.v.H., P.P.,
M.K.; revising the paper: all authors. All authors gave final ap-
proval of the version to be published.
Previous Posting: An earlier version of this article was previ-
ously posted to the Research Square preprint server: https://doi.
org/10.21203/rs.2.20192/v1.
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