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\begin{document}

\title{REPRODUCIBILITY OF EUCAPNIC VOLUNTARY HYPERVENTILATION FOR
EXERCISE-INDUCED BRONCHOSPASM DIAGNOSIS IN ASTHMATIC CHILDREN AND
ADOLESCENTS.}



\author[1]{José Rizzo}%
\author[1]{Edil Rodrigues Filho}%
\author[1]{Adriana  Gonçalves}%
\author[1]{Laienne  Albuquerque}%
\author[1]{Eduardo Albuquerque}%
\author[1]{Cláudio Albuquerque}%
\author[1]{Anderson Almeida}%
\author[1]{Decio Medeiros}%
\author[1]{Marco Correia Júnior}%
\affil[1]{Federal University of Pernambuco}%


\vspace{-1em}



  
  \date{May 17, 2024}


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\selectlanguage{english}
\begin{abstract}
BACKGROUND: Exercise-induced respiratory symptoms are frequently
reported by asthmatics and exercise-induced bronchospasm (EIB) is a
frequent cause that requires objective testing for diagnosis. Eucapnic
voluntary hyperventilation (EVH) is recommended as an exercise surrogate
stimulus for this purpose, but its short-term reproducibility is not yet
established in young asthmatics. OBJECTIVE: To evaluate the short-term
test-retest agreement and reproducibility of FEV1 changes after EVH in
young asthmatics. METHODS: Asthmatics aged between 10 and 20 years
underwent EVH for EIB diagnosis on two occasions 2-4 days apart at a
specialized university clinic. FEV1 was measured 5, 15 and 30 minutes
after EVH with a target ventilation rate 21 times baseline FEV1. EIB was
diagnosed as a decrease \textgreater{}10\% in FEV1 from baseline.
RESULTS: Twenty-six of 62 recruited individuals tested positive for EIB
on both visits (positive group) and 17 on one visit only (divergent
group); and 19 tested negative on both visits (negative group). The
overall agreement was 72.5\% (95\%CI 61.6\%, 83.6\%) and positive and
negative agreement was 41.9\% and 30.6\% respectively. Despite overall
low bias in FEV1 response between test days (0.87\%), the limits of
agreement were wide (+20.72\%). There were no differences in
pre-challenge FEV1 or achieved ventilation rate, between visits either
between groups (p=0.097 and p=0.461) or within groups, (p=0.828 and
p=0.780). No test was interrupted by symptoms and there were no safety
issues. CONCLUSIONS: More than one EVH test should be performed in young
asthmatics with a negative test to exclude EIB and minimize misdiagnosis
and mistreatment.%
\end{abstract}%



\sloppy


\textbf{Title Page}

\textbf{REPRODUCIBILITY OF EUCAPNIC VOLUNTARY HYPERVENTILATION FOR
EXERCISE-INDUCED BRONCHOSPASM DIAGNOSIS IN ASTHMATIC CHILDREN AND
ADOLESCENTS.}

Jose \selectlanguage{ngerman}Ângelo Rizzo MD, PhD\textsuperscript{1,2}, Edil de Albuquerque
Rodrigues Filho, Phys Ed, PhD\textsuperscript{1,2}, Adriana Velozo
Gonçalves, MD, MSc\textsuperscript{1}, Laienne Carla Barbosa De Barros
Albuquerque, PT, MSc\textsuperscript{1,3}, Eduardo Martins de
Albuquerque, Phys Ed, MSc\textsuperscript{1,3}, Cláudio Gonçalves de
Albuquerque, PT, MSc\textsuperscript{1,2}, Anderson Henrique Souza de
Almeida, Phys Ed, MSc\textsuperscript{1}, Decio Medeiros Peixoto, MD,
PhD\textsuperscript{1,2}, Marco Aurelio de Valois Correia Junior, PT,
PhD\textsuperscript{1,3,4}.

1 -- Center for allergy and clinical immunology research at Hospital das
Clínicas, Universidade Federal de Pernambuco, Recife, Brazil.

2 -- Infant and adolescent health post-graduation program, Universidade
Federal de Pernambuco, Recife, Pernambuco, Brazil.

3 -- Hebiatrics post-graduation program, Universidade de Pernambuco,
Recife, Pernambuco, Brazil.

4 --Physical Education post-graduation program, Universidade de
Pernambuco, Recife, Pernambuco, Brazil.

\textbf{Corresponding Author:} Edil de Albuquerque Rodrigues Filho.
Addres: R. Alto do Reservatório - Alto José Leal, Vitória de Santo Antão
-- PE, Centro Acadêmico de Vitória, Universidade Federal de Pernambuco.
Telephone number +5581-997484466. email address:
edil.albuquerque@ufpe.br

Financial support: This study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance
Code 001 and Hospital das Clínicas - Universidade Federal de Pernambuco.

Brazil.

Declaration of interest: The authors report no conflicts of interest

Word count for the Abstract: 248 words

Word count for the text: 3498 words

\textbf{REPRODUCIBILITY OF EUCAPNIC VOLUNTARY HYPERVENTILATION FOR
EXERCISE-INDUCED BRONCHOSPASM DIAGNOSIS IN ASTHMATIC CHILDREN AND
ADOLESCENTS.}

\textbf{ABSTRACT}

BACKGROUND: Exercise-induced respiratory symptoms are frequently
reported by asthmatics and exercise-induced bronchospasm (EIB) is a
frequent cause that requires objective testing for diagnosis. Eucapnic
voluntary hyperventilation (EVH) is recommended as an exercise surrogate
stimulus for this purpose, but its short-term reproducibility is not yet
established in young asthmatics. OBJECTIVE: To evaluate the short-term
test-retest agreement and reproducibility of FEV\textsubscript{1}changes
after EVH in young asthmatics. METHODS: Asthmatics aged between 10 and
20 years underwent EVH for EIB diagnosis on two occasions 2-4 days apart
at a specialized university clinic. FEV\textsubscript{1} was measured 5,
15 and 30 minutes after EVH with a target ventilation rate 21 times
baseline FEV\textsubscript{1}. EIB was diagnosed as a
decrease\emph{\textgreater{}} 10\% in FEV\textsubscript{1} from
baseline. RESULTS: Twenty-six of 62 recruited individuals tested
positive for EIB on both visits (positive group) and 17 on one visit
only (divergent group); and 19 tested negative on both visits (negative
group). The overall agreement was 72.5\% (95\%CI 61.6\%, 83.6\%) and
positive and negative agreement was 41.9\% and 30.6\% respectively.
Despite overall low bias in FEV\textsubscript{1} response between test
days (0.87\%), the limits of agreement were wide (\emph{+} 20.72\%).
There were no differences in pre-challenge FEV\textsubscript{1} or
achieved ventilation rate, between visits either between groups (p=0.097
and p=0.461) or within groups, (p=0.828 and p=0.780). No test was
interrupted by symptoms and there were no safety issues. CONCLUSIONS:
More than one EVH test should be performed in young asthmatics with a
negative test to exclude EIB and minimize misdiagnosis and mistreatment.

Key-words: asthma; adolescents; eucapnic voluntary hyperpnea;
exercise-induced bronchoconstriction; indirect bronchoprovocation
testing; reproducibility.













\section*{Abbreviations}

{\label{abbreviations}}

ACT = Asthma Control Test

AUC \textsubscript{0-30min =} area under the curve up to the thirtieth
minute

\section*{EIB = exercise-induced
bronchospasm}

{\label{eib-exercise-induced-bronchospasm}}

\section*{EIRS = Exercise-induced respiratory
symptoms}

{\label{eirs-exercise-induced-respiratory-symptoms}}

EVH = Eucapnic voluntary hyperventilation

FEV\textsubscript{1 =} Forced expiratory volume in the 1st second

















































\section*{Introduction}

{\label{introduction}}

Exercise-induced respiratory symptoms (EIRS) are frequently reported by
asthmatics, with rates ranging from 33\% in the Asia-Pacific area to
59\% in Central Eastern Europe\textsuperscript{1}. In Latin America,
this condition has been observed in 37\% of such
individuals\textsuperscript{2}. Many children and adolescents believe
their asthma is a factor that prevents them from participating in
physical activities and that this is an inherent feature of the disease.
This often brings with it the stigma of being less able and thus
restricts peer group activities and participation in sports and
games\textsuperscript{3,4,5}.

Although various conditions, including poor pulmonary function, poor
physical conditioning, exercise-induced laryngeal obstruction and
dysfunctional breathing \textsuperscript{6,7}, may be associated with
EIRS in children and adolescents with asthma, exercise induced
bronchospasm (EIB) is probably the most frequent and occurs in
approximately 50\%\textsuperscript{8} of them. EIB is defined as the
acute narrowing of the lower airways after exercise and may also
contribute significantly to avoidance of participation in physical
activities in children and young people with
asthma\textsuperscript{9,10}.

Diagnosis of EIB cannot be made solely on the basis of self-reported
respiratory symptoms, as it has poor predictive value compared to
objective tests\textsuperscript{11,12}. To avoid under- and
over-diagnosis and to enable correct treatment strategies to be adopted,
EIB must be established by measuring changes in lung function provoked
by exercise or a surrogate stimulus. The most common technique is serial
measurement of forced expiratory volume in the first second
(FEV\textsubscript{1}) before and after treadmill running. The criterion
for EIB diagnosis is a \emph{\textgreater{}} 10\% drop in
FEV\textsubscript{1} after the exercise challenge compared to the
baseline\textsuperscript{13}.

Eucapnic voluntary hyperventilation (EVH) has been recommended as a
surrogate stimulus for EIB diagnosis.\textsuperscript{13} It is
considered safe, enables better control of ventilation rates and
inspired air conditions, and dispenses with the need for physical
effort\textsuperscript{14,15,16,17}. The procedure follows the same
physiopathological pathways for EIB as treadmill running, namely
dehydration of the airway surface liquid of the bronchial
mucosa\textsuperscript{13}. In this method, the patient voluntarily
hyperventilates by breathing dry air enriched with 5\% CO2 (to avoid
respiratory alkalosis) at a pre-specified target ventilation
rate\textsuperscript{13}.

One important property of a diagnostic test is reproducibility and
knowledge of the inherent variability (agreement and repeatability) has
practical implications for evaluation of its clinical
usefulness\textsuperscript{18}. Anderson et al.\textsuperscript{19}found
a general agreement for EIB diagnosis of 76\% between two exercise
challenge tests carried out 1 to 4 days apart in 373 individuals
reporting signs or symptoms suggestive of asthma. Lower agreement (30\%)
between two tests separated by 1 to \textgreater{}35 days for EIB
diagnosis was found in a study of asthmatic adults when the diagnostic
cutoff was a 15\% drop in FEV\textsubscript{1}\textsuperscript{20}.

The few studies that examined the reproducibility of EVH for EIB
diagnosis were conducted in adult athletes and included a small number
of individuals with asthma\textsuperscript{21,22} or compared different
EVH provocation protocols\textsuperscript{23}. One study reported the
results of repeated EVH tests in a small number of non-athlete adult
males with asthma (eight individuals) and found a 100\% agreement
between challenge responses, obtained seven days apart or with an
interval of 35 days\textsuperscript{24}. We were not able to find
studies of the short-term reproducibility of
FEV\textsubscript{1}response in asthmatic children and adolescents after
EVH challenge.

The aim of the present study was to evaluate the short-term test-retest
agreement and reproducibility (repeatability) of
FEV\textsubscript{1}changes after EVH in children and adolescents with
medically diagnosed asthma.

\textbf{Methods}

The study was approved by the Institutional Ethics Committee and signed
informed consent was obtained from parents/guardians along with the
assent of children/adolescents.

Study population

Patients aged 10 to 20 years were recruited from the pediatric allergy
and immunology clinic of the Hospital das Clínicas of the Universidade
Federal de Pernambuco, in Recife -- Brazil between 2017 and 2019. All
had received a specialist diagnosis of asthma based on GINA
criteria\textsuperscript{25}.

Individuals were excluded if they had a recent history (less than 6
weeks) of asthma exacerbation or acute respiratory infection symptoms,
were regularly using inhaled steroids, were smokers or ex-smokers, had
any other known pulmonary diseases, were unable to perform adequate
forced expiratory maneuvers, or had a baseline
FEV\textsubscript{1}\textless{} 60\% of predicted FEV\textsubscript{1}.

Procedures

All subjects were required to attend the laboratory on two occasions
separated by a period of 2 to 4 days at a similar time of day (\emph{+}2
hours), in the morning. On the first visit, anthropometric measurements
were taken and patients responded to the Asthma Control Test (ACT)
(Portuguese/Brazilian version)\textsuperscript{26}. They then underwent
spirometry to determine baseline
FEV\textsubscript{1}\textsuperscript{27} using a daily calibrated
spirometer (KitMicro Cosmed, Rome, Italy). The best of three acceptable
maneuvers was chosen. If the value was \emph{\textgreater{}} 60\% of
that predicted (for the Brazilian population\textsuperscript{28}),
individuals proceeded to the EVH challenge. On the second visit,
baseline FEV\textsubscript{1} was measured and, if the best value lay
within 15\% of that of the first day (but higher than 60\% of
predicted), patients proceeded to the EVH challenge. The laboratory room
temperature was controlled and air humidity was measured using a thermo
hygrometer (Incoterm, Brazil).

Patients were instructed to abstain from caffeine and exercise on test
days and from using short- and long-acting beta-2 agonists for 12 and 48
hours, respectively. It was recommended that patients not use inhaled
corticosteroids on the test day\textsuperscript{13}.

Eucapnic voluntary hyperpnea and spirometry

Patients breathed a mixture of dry room-temperature air with 5\% added
carbon dioxide (CO2) (White-Martins, Recife, PE - Brazil) collected in a
Douglas balloon, through the mouth, with nose clipped using a one-way
low-resistance valve (Laerdal, Copenhagen -Denmark). The test lasted 6
min and the target ventilation per minute was set at 21 times that of
the personal baseline FEV\textsubscript{1} (equivalent to 60\% of
predicted maximal voluntary ventilation)\textsuperscript{13}. The
ventilation rate per minute was monitored using an analog ventilometer
(Wright Mark 8 NSPIRE Health, Colorado - USA) and subjects were coached
every 30 seconds to maintain the target ventilation level. The
ventilation rate was recorded every minute for six minutes and the mean
expressed as a percentage of the calculated target ventilation.
Spirometry for FEV\textsubscript{1} measurement was performed in
duplicate 5, 15 and 30 minutes after EVH and the highest value recorded.
The \% drop in FEV\textsubscript{1} was calculated using the formula
(baseline FEV\textsubscript{1}-- post-EVH FEV\textsubscript{1}/baseline
FEV\textsubscript{1}.100) and the maximum decrease in
FEV\textsubscript{1} at any of the three points in time was recorded
(FEV\textsubscript{1}fallmax\%)\textsuperscript{13}. Subjects were
considered positive for EIB diagnosis if a decrease
of\emph{\textgreater{}} 10\% in FEV\textsubscript{1} was observed at any
evaluation time-point on at least one of the test days.

Individuals were divided into three groups: those with a
FEV\textsubscript{1}fallmax\% \emph{\textgreater{}} 10\% on both test
days, those with FEV\textsubscript{1}fallmax\% \emph{\textgreater{}}
10\% on one day only (divergent group) and those with
FEV\textsubscript{1}fallmax\% \textless{}10\% on both test days. The
severity of EIB was graded as mild, moderate, or severe if the
percentage decrease in FEV\textsubscript{1} from the pre-exercise level
was \emph{\textgreater{}} 10\%\textless{} 25\%,\emph{\textgreater{}}
25\%\textless{} 50\%, and\emph{\textgreater{}} 50\%,
respectively\textsuperscript{13}.

Statistical analysis

Data were processed and analyzed using Statistical Package for the
Social Sciences (SPSS), version 20.0 and figures produced using
GraphPadPrism® version 6.00 for Windows. Normally distributed data
(Kolmogorov-Smirnov) are expressed as means \emph{+} SD and/or 95\%
confidence interval (CI) and non-normal data as median and interquartile
range (IQR). Agreement was evaluated as a binary outcome considering the
proportion of positive and negative response on both tests (proportion
of agreement). Reproducibility (repeatability) and limits of agreement
(LOAs) were assessed for FEV\textsubscript{1}fallmax\% and
AUC\textsubscript{0-30min} using the method described by Bland \&
Altman\textsuperscript{29}. Individuals who tested positive on both days
with a FEV\textsubscript{1}falmax\% \emph{\textgreater{}} 15\% and those
with \emph{\textgreater{}} 20\% were also analyzed separately for bias
and LOAs in these sub-groups. The area under the curve up to the
thirtieth minute (AUC \textsubscript{0-30min}) was calculated by the
trapezoidal method using the percentage of FEV\textsubscript{1}fallmax\%
at each point in the evaluation. For the calculation of
AUC\textsubscript{0-30min}, positive variations in
FEV\textsubscript{1}after EVH were considered zero. Multivariate
analysis of variance (MANOVA) was used to compare baseline
FEV\textsubscript{1} as a percentage of predicted and the achieved
ventilation rate as a percentage of the target calculated by group (with
and without BIE) and visit. The Kruskal-Wallis test was performed to
assess the differences in ACT between the groups. The intra-class
correlation coefficient (ICC) for the FEV\textsubscript{1}falmax\% and
AUC\textsubscript{0-30min} was calculated using a two-way mixed-effect
model with the mean single measurement reported to evaluate relative
reliability. The alpha error probability was set at 0.05.

\textbf{Results}

Baseline characteristics

Sixty-two asthmatic children and adolescents completed the study. Seven
did not attend on the second day because of transport difficulties and
were excluded. All individuals were able to complete the six-minute EVH
and there was no need to interrupt the 30-minute FEV1 evaluation because
of respiratory symptoms.

No patient was excluded because of a low predicted
FEV\textsubscript{1}level. Twelve were prescribed a low-dose
(200mcg/day) continuous-use corticosteroid inhaler (beclomethasone,
which is distributed free of charge by the government) but none were
using it regularly and none had used it for at least one week before
testing. No patient was using long-acting beta-2 agonists and all had
albuterol for recovery. General data are presented in Table 1.

Mean laboratory temperature was 25.5\textsuperscript{o}C (\emph{+}
2.8\textsuperscript{o}C) and relative humidity 58.7\% (\emph{+} 6.2\%),
with no differences between test days (p\textgreater{}0.05).

Reproducibility and agreement

Twenty-six of the 62 patients responded positively for EIB after EVH on
both days (i.e., had a
FEV\textsubscript{1}fallmax\%\emph{\textgreater{}} 10\%), 17 on one day
only (5 on the first day and 12 on the second), and 19 responded
negatively on both days. The overall agreement was 72.5\% (95\% CI
61.6\%, 83.6\%) and the positive and negative agreement proportions were
41.9\% and 30.6\% respectively.

No difference was observed in mean FEV\textsubscript{1}fallmax\% after
EVH between visits for the group as a whole, with low bias (mean
difference between visits for FEV\textsubscript{1}fallmax\%) but with
wide LOAs. The same was found for the AUC\textsubscript{0-30min} (Table
2 and Figure 1). Seventeen out of 26 patients with a positive response
on both test days (65.4\%) experienced a decrease in
FEV\textsubscript{1} \emph{\textgreater{}} 15\%, and, in 10 of these,
the reduction was \emph{\textgreater{}} 20\%. In this sub-group of
individuals, bias was also small and LOAs were wide for both
FEV\textsubscript{1}falmax\% and AUC\textsubscript{0-30min} (Table 2).
The mean FEV\textsubscript{1}fallmax\% in those individuals testing
positive for EIB on one visit only (divergent group) was 17.7\%\emph{+}
13.5\% on the positive day (either visit one or two) and was
statistically different from that observed in those individuals testing
positive for EIB on both days (p = 0.016 for visit 1 / p = 0.021 for
visit 2).

For the group as a whole, the intra-class correlation coefficient (ICC)
for the FEV\textsubscript{1}fallmax\% between the two visits was 0.854
(95\% CI 0.758, 0.912; p\textless{}0.001) and was 0.858 (95\% CI 0.764,
0.915; p\textless{}0.001) for AUC\textsubscript{0-30min}.

There was a significant correlation between
FEV\textsubscript{1}fallmax\% and AUC\textsubscript{0-30min} for both
visits (visit 1: r = 0.91, p \textless{} 0.001 and visit 2: r = 0.89, p
\textless{} 0.001).

Confounding factors

Potential differences in baseline FEV\textsubscript{1} (as a percentage
of predicted) and achieved ventilation rate (as a percentage of the
calculated target) between visits were considered confounding factors
that could interfere with the FEV\textsubscript{1} response after EVH.
As shown in Table 3, no differences between visits were observed in
these two parameters either between groups or within groups. A more
detailed overview of the individual target ventilation rates achieved on
both visits for each group is provided in Figure 2.

Baseline asthma control status measured using the ACT score was also
considered a potentially confounding variable but showed no difference
between groups (Table 3). There were no differences either between
groups in terms of age and BMI (p=0.624 and p=.0957, respectively -
Kruskal-Wallis) or sex (p=.0738 - Chi-square).

Severity of EIB and recovery

During visit one, FEV\textsubscript{1} did not return spontaneously to
baseline levels by the thirtieth minute in 15 of the individuals testing
positive on both days (n=26), and, during visit two, in 14. In those
testing positive on one day only, this occurred in 6/17. These
individuals were given 400mcg inhaled albuterol and
FEV\textsubscript{1}returned to baseline values (within 10\%) in all of
these. Among those testing positive on both days, the severity of
FEV\textsubscript{1}fallmax\% was graded as mild on Visits 1 and 2 in 16
and 13, respectively, as moderate in 8 and 11, and as severe in the same
two individuals on both days. Fourteen of the individuals testing
positive on one visit only had a mild FEV\textsubscript{1}fallmax\%
response and three had a moderate response. There was no need to
interrupt the FEV1 measurements after EVH due to respiratory complaints
or oxygen desaturation below 94\%.

\textbf{Discussion}

Knowledge of the reproducibility of a test is paramount for
understanding its clinical utility for diagnosis or evaluation of
changes over time and responses to therapeutic
interventions\textsuperscript{18}. In a group of 62 children and
adolescents with asthma, we found the EVH challenge test to have a
short-term FEV\textsubscript{1} reproducibility response of 72.5\% for
EIB diagnosis, with positive and negative agreement of 41.9\% and
30.6\%, respectively. It should be noted that, in 12 patients, EIB
occurred only on the second challenge. This is relevant for the
differential diagnosis of respiratory symptoms upon exercise in young
asthmatics, in whom EIB is one of the most common but not the only
triggering factor, with consequences for the individual patient and the
choice and monitoring of treatment options\textsuperscript{6,7,30,31}.
Our study fills a gap in the literature, as published studies on the
reproducibility of the EVH have been conducted only in
athletes\textsuperscript{21,22} or in small numbers of asthmatic
adults\textsuperscript{23,24}. The presence of EIB is defined by a
decrease in FEV\textsubscript{1} \emph{\textgreater{}} 10\% from
baseline, either for exercise or EVH challenges\textsuperscript{13,17}.
For research purposes, the dimensionless area under the
FEV\textsubscript{1}fallmax\% curve and ICC may contribute to general
understanding of the bronchial response and repeatability but are
difficult to interpret on clinical grounds. Despite a high ICC and low
bias, we found wide limits of agreement (LOAs), both for
FEV\textsubscript{1}fallmax\% and AUC\textsubscript{0-30min} (Figure 1).
As shown in Table 2, the overall LOAs were similar among those
individuals with a positive response on two visits, irrespective of the
magnitude of the FEV\textsubscript{1}fallmax\%. In an earlier study
comparing EIB response between EVH and treadmill running challenges in a
similar population, we also observed wide LOAs between tests
(\emph{+}19.7\%)\textsuperscript{32}.

The overall LOAs are wider than those found by Anderson et
al.\textsuperscript{19} in exercise tests in their group of children
(\emph{+} 13.4\%). This may be explained by the lower rate of negative
agreement found in our study (30.6\% compared to 56.8\%) and may be a
consequence of the difference in study populations, as Anderson's study
group had symptoms compatible with asthma, but no medical diagnosis.
Despite these differences, the overall agreement for EIB diagnosis in
our study varies little from that observed by these authors, who found
76\% agreement between two exercise tests conducted one to four days
apart. Price et al.\textsuperscript{21} found a 75\% agreement between
EIB responses to EVH tests on two occasions (14-21 days apart) in 32
adult athletes (19\% with asthma) with a high proportion of negative
agreement (55\%). These authors considered the limits of agreement to be
wide (\emph{+} 10.1\%), although they were approximately 50\% lower than
in our study. This too may be explained by differences in study
population.

Our population comprised young individuals receiving an asthma diagnosis
at a tertiary university specialty care facility, most (95\%) classified
as having controlled or partially controlled asthma, with a score
\textgreater{}18 on the ACT (Brazilian version\textsuperscript{26}).
There were no differences in asthma control score between EVH response
groups (Table 3). No patient was regularly using inhaled corticosteroids
capable of interfering with the EIB response\textsuperscript{33}.

The recommended target ventilation rate during the six-minute EVH test
for elite athletes is 85\% of maximum voluntary ventilation (MVV), i.e.,
30 times the baseline FEV\textsubscript{1}. For non-athletes, 60\% of
MVV is considered adequate (21 times baseline
FEV\textsubscript{1})\textsuperscript{13,34}. We adopted this as the
target ventilation rate to be achieved during the EVH challenge.
Overall, there were no differences in the mean ventilation rate achieved
between visits or between the three groups (Table 3). Most individuals
(48/62) were able to achieve more than 80\% of the target ventilation,
but fourteen (22.5\%) achieved values between 60\% and 80\%. This may be
a weakness of our study. Detailed examination of these individuals
reveals that four and eight were in the positive and negative EIB groups
on both visits respectively, and two tested positive on one day only (p
= 0.119, Chi Square).

No correlation was found between target ventilation and the
FEV\textsubscript{1}falmax\% for any visit (Visit 1: r = -0.20, p =
0.110, Visit 2: r = -0.12, p = 0.358 -- Spearman's rho). These findings
are consistent with those of Brummel et al.\textsuperscript{16} and
Stadelmann et al.\textsuperscript{22}, who found the ventilation rate
not to be related to FEV\textsubscript{1}fallmax\% after EVH. More
studies need to be conducted to evaluate this relationship. Unlike these
authors, we found no differences in ventilation levels achieved during
EVH between males and females (p=0.430).

Published guidelines recommend a baseline FEV\textsubscript{1} of over
75\% of predicted as a safety measure to avoid severe bronchospastic
responses\textsuperscript{35,36}. Brannan and
Kippelen\textsuperscript{34} recommend that individuals known to be
asthmatic be excluded from performing EVH. Parsons et
all.\textsuperscript{13} do not provide specific safety recommendations.
The experience in our laboratory, as reported by other
publications\textsuperscript{14,16,17,23,} is that a safe EVH test can
be conducted in asthmatics with a baseline FEV\textsubscript{1} as low
as 60\% of predicted. Of course, the test must be conducted by trained
personnel with resources available for a possible emergency event. On
Visits 1 and 2, 11 and 14 patients respectively had baseline
FEV\textsubscript{1} \textless{}75\% but \textgreater{}60\% of
predicted; only one presented a severe EVH response
(FEV\textsubscript{1}fallmax\% \emph{\textgreater{}} 50\%) but had no
severe respiratory symptoms or hemoglobin oxygen saturation below 94\%.

Different from Hurwitz et al.\textsuperscript{37}, who observed a weak
but statistically significant correlation between baseline
FEV\textsubscript{1} and the FEV\textsubscript{1}fallmax\%, we were
unable to detect such a correlation on either of the two visits (Visit 1
r = 0.165, p = 0.201; Visit 2 r =0.178, p =0.166. Spearman's rho).

The limits of agreement between repeated tests allow us to evaluate the
magnitude of response that can be considered to lie outside expected
variations in the parameter\textsuperscript{29}. For a treatment to be
considered of benefit in individuals with a FEV\textsubscript{1}falmax\%
greater than 20\%, using the LOAs for this study population with a mean
drop of 37.5\%, the FEV\textsubscript{1}fallmax\% after administration
of the drug would need to be less than 18\%. On the other hand, the
narrow LOAs observed for those individuals with negative response on
both visits (Table 2) agree with the results of Burman et
all.\textsuperscript{17} that found a 10\% fall in
FEV\textsubscript{1}as a good cutoff value for EIB diagnosis after EVH.

Bias in a test repeatability evaluation may be related to the execution
of the test itself (measurement error), changes in environmental
conditions or in individual factors, such as medication use, severity of
disease, and exposure to triggering factors. The patients in our study
were a homogeneous group in terms of asthma diagnosis, disease control
level and medication use. The EVH tests were repeated within a short
period of time to avoid any time-related changes in airway
responsiveness, disease conditions or environmental exposure. No
statistical differences were observed in baseline FEV\textsubscript{1}\%
of predicted between visits. Although patients were coached to achieve
the target ventilation rate (60\% of MVV), some did not reach this
level. However, comparison of individuals with divergent EIB responses
to EVH on separate days revealed no differences in the mean ventilation
rates achieved suggesting that this relationship deserves more
investigation. Although undetected factors may have interfered with the
short-term reproducibility of response to the EVH stimulus in 17
patients that presented a different EIB response on each visit, our
previous findings\textsuperscript{33}, like those of Price et
al.\textsuperscript{21} and Anderson et al.\textsuperscript{19}, suggest
that there may be an inherent individual variability in bronchial
response to EVH or exercise test, even between short term evaluations.

Our study was conducted in young asthmatics, most with well controlled
disease and with a short interval between tests. The findings cannot,
thus, be extrapolated to other populations or to larger intervals
between tests, for which specifically designed studies should be
conducted. It remains intriguing, however, that the same bronchial
stimulus elicits different bronchial responses in different individuals
with the same condition and, in the same individuals, on different
occasions.

In conclusion, it is clear that, for the moment, there are no diagnostic
tests that can be considered a ``gold standard'' for EIB diagnosis,
either in athletes\textsuperscript{38} or in asthmatic
individuals\textsuperscript{19}. Without a repeated EVH test, 28\% of
our patients with EIB diagnosis would have been missed. There is a need
for cautious interpretation of a negative EVH challenge for EIB
diagnosis and two or more tests should be performed in those patients in
whom EIB is to be excluded. Keen clinical judgment needs to be
exercised, especially in cases of diagnosis and therapeutic intervention
in patients with exercise-related respiratory complaints and mild or no
decrease in FEV\textsubscript{1} after EVH challenge.

Table 1. Baseline characteristics of patients\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{N} & 62\tabularnewline
\midrule
\endhead
\textbf{Sex} &\tabularnewline
Male (\%) & 25 (40\%)\tabularnewline
\textbf{Age (Years)} &\tabularnewline
Mean (SD) & 13.2 (2.7)\tabularnewline
Range & 10 -- 20\tabularnewline
\textbf{Height (cm)} &\tabularnewline
Mean (SD) & 152.8 (11)\tabularnewline
Range & 134 - 181\tabularnewline
\textbf{Weight (Kg)} &\tabularnewline
Mean (SD) & 47.9 (13.2)\tabularnewline
Range & 24.7 -- 72.0\tabularnewline
\textbf{BMI} &\tabularnewline
Mean (SD) & 20.2 (4.0)\tabularnewline
Range & 13.6 - 29.9\tabularnewline
\textbf{FEV\textsubscript{1} (L.sec\textsuperscript{-1})}
&\tabularnewline
Mean (SD) & 2.44 (0.72)\tabularnewline
Range & 1.19 - 4.18\tabularnewline
\textbf{FEV\textsubscript{1} \% Pred} &\tabularnewline
Mean (SD) & 89.0 (16.6)\tabularnewline
Range & 60.5 -- 124.0\tabularnewline
\textbf{ACT} &\tabularnewline
Median & 23\tabularnewline
IQR & 20 -- 25\tabularnewline
Range & 16 -- 25\tabularnewline
\bottomrule
\end{longtable}

BMI- Body mass index. FEV\textsubscript{1} = Forced expiratory

volume in the first second. ACT = Asthma control

test. SD = Standard deviation. IQR = Interquartile range.



\textbf{Table 2 - Mean highest decrease of FEV\textsubscript{1} as
percentage of basal values and area under the curve of
FEV\textsubscript{1} decrease from basal (as percentage) between zero
and the thirtieth minute of evaluation after EVH for both visits and
bias and limits of agreement (LOAs) for each category.}\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllll@{}}
\toprule
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
FEV\textsubscript{1}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
FEV\textsubscript{1}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
FEV\textsubscript{1}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
FEV\textsubscript{1}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
FEV\textsubscript{1}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
AUC\textsubscript{0-30min}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
AUC\textsubscript{0-30min}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
AUC\textsubscript{0-30min}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
AUC\textsubscript{0-30min}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
AUC\textsubscript{0-30min}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
N\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Mean FEV\textsubscript{1} fallmax\% (SD)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Mean FEV\textsubscript{1} fallmax\% (SD)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
p\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Bias* (SD)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
LOAs\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Mean AUC\textsubscript{0-30min} (SD)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Mean AUC\textsubscript{0-30min} (SD)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
p\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
Bias** (SD)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
LOAs\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Visit 1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Visit 2\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Visit 1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Visit 2\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
All patients\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
62\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
14.91 (14.50)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
15.77 (13.71)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
0.520\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
0.87 (10.57)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{+} 21.14\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
327.18 (295.26)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
291.72 (328.42)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
0.165\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
19.90 (198.68)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{+} 397.36\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
2 tests fal \emph{\textgreater{}}10\%\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
26\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
26.68 (13.46)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
26.38 (13.49)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
0.701\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
0.29 (10.73)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{+} 22.06\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
516.00 (273.00)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
521.06 (285.13)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
0.604\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
5.05 (218.92)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{+} 450.09\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
2 tests fall \emph{\textgreater{}}15\%\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
17\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
32.47 (13.07)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
31.44 (13.12)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
0.717\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
1.03 (11.5)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{+} 24.26\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
619.22 (274.99)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
632.41 (262.02)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
0.786\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
13.19 (210.82)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{+} 444.83\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
2 tests fall \emph{\textgreater{}}20\%\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
10\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
37.46 (12.23)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
38.65 (11.92)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
0.666\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
1.18 (8.37)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{+} 19.08\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
726.21 (291.84)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
751.64 (213.34)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
0.763\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
25.43 (176.47)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{+} 393.17\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
1 test fall \emph{\textgreater{}} 10\%\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
17\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
10.74 (8.94)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
12.63 (7.84)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
0.607\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
1.90 (14.91)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{+} 31.46\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
211.61 (225.96)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
173.19 (74.88)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
0.562\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
38.42 (267.50)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{+} 564.42\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
2 tests fall \textless{}10\%\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
19\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
2.53 (3.43)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
4.07 (3.66)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
0.061\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
1.54 (3.35)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{+} 7.01\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
82.66 (64.68)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
99.84 (62.94)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
0.083\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
17.18 (40.76)\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{+} 85.31\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

LOAs = Limits of agreement. AUC\textsubscript{0-30min} = Area under the
curve up to the thirtieth minute. Mean FEV\textsubscript{1}fallmax\% =
mean percentage maximum decrease in FEV\textsubscript{1} compared to
baseline. Bias* = Mean difference (SD) in FEV\textsubscript{1}fallmax\%
between 2 days. Bias** = Mean difference in
AUC\textsubscript{0-30min}(SD) between 2 days.

\textbf{Table 3 --Analysis of associated factors possibly influencing
FEV\textsubscript{1} response after eucapnic voluntary hyperventilation}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllllll@{}}
\toprule
\begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
All (n=62)\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
All (n=62)\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Groups\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Groups\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Groups\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Groups\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Groups\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Groups\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
FEV\textsubscript{1} decrease \emph{\textgreater{}} 10\% on 2 days
(n=26)\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
FEV\textsubscript{1} decrease \emph{\textgreater{}} 10\% on 2 days
(n=26)\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
FEV\textsubscript{1} decrease \emph{\textgreater{}} 10\% on 1 day
(n=17)\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
FEV\textsubscript{1} decrease \emph{\textgreater{}} 10\% on 1 day
(n=17)\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
FEV\textsubscript{1} decrease \textless{} 10\% on 2 days (n=19)\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
FEV\textsubscript{1} decrease \textless{} 10\% on 2 days (n=19)\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
MANOVA analysis\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
MANOVA analysis\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
MANOVA analysis\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
MANOVA analysis\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
V 1\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
V 2\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
V 1\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
V 2\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
V 1\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
V 2\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
V 1\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
V 2\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
Visits\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
Visits\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
Groups\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
Groups\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{Basal FEV\textsubscript{1} \% pred}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
F\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
p\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
F\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
p\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
Mean\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
88.9\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
87.94\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
88.18\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
86.48\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
85.27\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
84.61\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
92.03\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
92.56\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0.047\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0.8285\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
2.379\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0.0971\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
(SD)\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
15.92\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
14.9\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
16.01\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
14.79\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
15.42\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
18.05\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
13.50\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
14.48\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{Achieved ventilation} \textbf{as \% of target calculated}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
Mean\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
96.45\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
94.81\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
99.73\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
98.48\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
90.88\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
96.81\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
92.47\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
94.55\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0.3017\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0.5839\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0.7801\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0.4607\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
(SD)\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
24.62\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
20.64\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
23.93\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
20.12\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
14.58\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
25.75\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
25.32\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
23.13\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{ACT* V1}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
Kruskal--Wallis test\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
Kruskal--Wallis test\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
Kruskal--Wallis test\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
Kruskal--Wallis test\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
Median\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
23\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{-}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
23\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
22\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
24\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{-}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
p\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
p\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
IQR\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
20-25\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{-}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
19.5-25\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
19.5-24\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
22-25\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{-}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0.2450\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0.2450\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

FEV\textsubscript{1} \% pred. = Forced expiratory volume in the first
second expressed as percentage of predicted value. ACT = Asthma control
test. V = Visit. MANOVA= multivariate analysis of variance. SD= Stand
deviation. IQR = interquartile range

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/397288/articles/510221-reproducibility-of-eucapnic-voluntary-hyperventilation-for-exercise-induced-bronchospasm-diagnosis-in-asthmatic-children-and-adolescents}

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/397288/articles/510221-reproducibility-of-eucapnic-voluntary-hyperventilation-for-exercise-induced-bronchospasm-diagnosis-in-asthmatic-children-and-adolescents}

\textbf{Figure 1. Bland-Altman plot of the maximum decrease in
FEV\textsubscript{1} (upper figure) and area under the curve from
baseline to thirtieth minute (lower figure) after EVH, comparing visits.
Filled circles indicate individuals with EIB on both visits, empty
circles individuals with EIB on one visit only (divergent response), and
split circles individuals with no EIB.}

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/397288/articles/510221-reproducibility-of-eucapnic-voluntary-hyperventilation-for-exercise-induced-bronchospasm-diagnosis-in-asthmatic-children-and-adolescents}

\textbf{Figure 2. Ventilation achieved (as \% of calculated) on Visits 1
and 2 (Day 1, Day 2). A = individuals with EIB on both visits, B =
individuals without EIB, C = individuals with EIB on one visit only
(divergent response).}

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\selectlanguage{english}
\FloatBarrier
\end{document}

