ArticlePDF Available

Investigation of everyday influencing factors on the variability of exhaled breath profiles in healthy subjects


Abstract and Figures

Introduction: The human breath is an accurate but complex read-out of many physiological processes in the organism that can be monitored via volatile organic compounds (VOCs) in the exhaled air. However, there are many confounding variables that limit the transfer and application of breath analysis to become a clinical procedure. Method: This work aims to establish a systematic procedure for sampling and characterization of various everyday influences of healthy subjects using proton transfer reaction-mass spectrometry (PTR-MS). In order to limit the influencing factors on the breath profile, a standard analysis procedure for sampling and evaluation of the exhaled breath samples was developed. The correlations between the selected experimental conditions and the resulting VOC profiles were investigated using a non-parametric Wilcoxon rank sum test. Results: In addition to the relevant influence of methodological experimental parameters, interesting insights into the effect of everyday factors on the exhalat gas were obtained and discussed. Furthermore, subject and condition-specific differences were found in the exhaled air of male and female subjects. Conclusion: With a more robust, standardized and reproducible breath sampling protocol, breath analysis is a promising non-invasive tool towards a system-wide understanding and personalized diagnosis and treatment of a wide range of diseases.
Content may be subject to copyright.
Melanie Fachet*, Simon Lowitzki, Marie-Louise Reckzeh, Thorsten Walles, and Christoph
Investigation of everyday influencing factors
on the variability of exhaled breath profiles in
healthy subjects
Introduction: The human breath is an accurate but complex
read-out of many physiological processes in the organism that
can be monitored via volatile organic compounds (VOCs) in
the exhaled air. However, there are many confounding vari-
ables that limit the transfer and application of breath analysis
to become a clinical procedure.
Method: This work aims to establish a systematic procedure
for sampling and characterization of various everyday influ-
ences of healthy subjects using proton transfer reaction-mass
spectrometry (PTR-MS). In order to limit the influencing fac-
tors on the breath profile, a standard analysis procedure for
sampling and evaluation of the exhaled breath samples was
developed. The correlations between the selected experimen-
tal conditions and the resulting VOC profiles were investigated
using a non-parametric Wilcoxon rank sum test.
Results: In addition to the relevant influence of methodolog-
ical experimental parameters, interesting insights into the ef-
fect of everyday factors on the exhalat gas were obtained and
discussed. Furthermore, subject and condition-specific differ-
ences were found in the exhaled air of male and female sub-
Conclusion: With a more robust, standardized and repro-
ducible breath sampling protocol, breath analysis is a promis-
ing non-invasive tool towards a system-wide understanding
and personalized diagnosis and treatment of a wide range of
Keywords: Breath gas analysis, Breathomics, Proton-
transfer-reaction mass spectrometry, Subject variability, Ev-
eryday influencing factors
*Corresponding author: Melanie Fachet, Simon Lowitzki,
Christoph Hoeschen, Institute for Medical Technology, Chair of
Medical Systems Technology, Otto von Guericke University
Magdeburg, Universitätsplatz 2, 39106 Magdeburg, Germany.
Marie-Louise Reckzeh, Thorsten Walles, University Clinic for
Cardiac and Thoracic Surgery, Otto von Guericke University
Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany.
1 Introduction
In the last decades, the analysis of volatile organic compounds
(VOCs) in the exhaled human breath has been studied and
developed as a promising technique to identify biomarkers
for the diagnosis and monitoring of various diseases. One of
the main advantages of this procedure is its non-invasive ap-
proach. This is particularly important for populations such as
children and elderly people, and for diseases whose current
standard diagnoses use invasive techniques such as biopsies
and bronchoscopies or imaging methods based on ionizing ra-
diation. Despite of the ongoing advances in this research field,
breath gas analysis is not yet a routine clinical tool. Standard
clinical procedures have not yet been sufficiently established
and validated, especially when it comes to identifying suitable
biomarkers and setting comparative values for healthy volun-
teers [10].
Recent advancements in breath research have led to the iden-
tification of biomarkers in a wide range of diseases such
as lung and breast cancer, COPD, asthma, diabetes, dis-
eases of the skin barrier and many more [2, 4, 6, 9]. Var-
ious methods are available for the measurement of VOCs,
which vary in their detection sensitivity and analysis speed e.g.
gas chromatography-mass spectrometry (GC-MS), electronic
nose, proton transfer reaction-mass spectrometry (PTR-MS),
ion mobility spectrometry (IMS), chemiluminescence or opti-
cal absorption detection techniques [11].
The work presented in this paper uses PTR-MS, which of-
fers the advantages of a rapid response, a soft chemical ion-
ization principle along with the possibility for an absolute
quantification and a high sensitivity down to a parts per tril-
lion (ppt) level [11]. PTR-MS is based on a soft chemical
ionization, where a hydronium ion (H3O+) is used to charge
VOC molecules Rfor proton affinities higher than for water
molecules [1].
It has been shown that a breath sample contains more than
3.500 different VOCs, mostly in the picomolar range. The gas
exchange between the blood system and the external environ-
ment can be monitored in the human breath. This process of
alveolar gas exchange with the blood facilitates oxygen up-
take and releases by-products of metabolic reactions such as
cdbme_2022_8_2.pdf 261 8/29/2022 5:45:49 PM
Current Directions in Biomedical Engineering 2022;8(2): 261-264
Open Access. © 2022 The Author(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License.
M. Fachet et al., Breath gas variability in health volunteers
exhaled breath gas volatiles such as methanol, ethanol, ac-
etaldehyde, acetone and isoprene [8]. The specific composi-
tion of the individual breath pattern is influenced by the sub-
ject’s physiological situation, the lifestyle and state of health
[8]. However, there is currently a lack of standardization in
breath sampling, data acquisition and analysis for the process-
ing of PTR-MS data from clinical study cohorts that are suited
for biomarker identification.
The work presented in this paper aims to investigate the influ-
ence of everyday lifestyle factors and the volatile biomarker
profiles in healthy subjects affecting the reliability, repro-
ducibility and suitability of breath gas analysis for the design
of clinical studies. In this setting, several everyday influenc-
ing factors such as getting up, brushing teeth and food up-
take were investigated for its variability in the VOC abundance
along with possible interferences from ambient air samples.
In this study, the influence of different everyday factors was
determined by measuring the complete range of VOC mass-
to-charge ratios between m/z 20 to m/z 200 [8]. It is impor-
tant to determine which of the various sampling parameters
has a larger influence on the VOC profile and should there-
fore be avoided in clinical studies to minimize inter- and intra-
individual variability of confounding factors.
2 Material and Methods
2.1 Study population
The healthy subject population consisted of 13 voluntary re-
searchers (7 male and 6 female subjects) from the Chair of
Medical Systems Technology and the University Clinic for
Cardiac and Thoracic Surgery. The informed consent was ob-
tained from the study participants and the study was approved
by the institutional ethics committees on human research of
the Otto-von-Guericke University Magdeburg (vote 194/20).
2.2 Breath gas sampling
Breath gas samples were collected in 3 l Tedlar bags. In or-
der to ensure a minimum level of contaminations in the reused
bags, the bags were purged with nitrogen (99.5% purity) twice
and a low background VOC level was verified by additional
measurements. The exhalation volume of 2-2.5 l was typically
reached after 20–30 s containing a mixed fraction of the sub-
ject’s exhaled breath and the samples were subsequently mea-
sured within 2 h after breath collection. Ambient air samples
were taken from laboratories and office rooms where the study
subjects were located in.
2.3 Measurement of samples with
The breath gas analysis was conducted using a commercial
standard PTR-MS with Time-of-Flight (TOF) mass detec-
tor (PTR-TOF 2000, Ionicon Analytik, Innsbruck, Austria),
which allows very sensitive offline and online measurements
in the low ppb to ppt range. The breath sampling was per-
formed as previously described by [1] and [8]. Briefly, the
PTR-MS measurements were performed with a drift tube pres-
sure of approximately 2.3 mbar. The VOC masses were ana-
lyzed in consecutive scans from a mass-to-charge ratio ranging
from m/z 20 to 200.
2.4 Statistical analysis
The measured VOC profiles of the healthy volunteers were
evaluated using the statistical toolbox in MATLAB (Math-
Works, Version R2021a). Descriptive measures included the
median and interquartile range (IQR) for the selected breath
volatiles. Furthermore, a non-parametric test Wilcoxon rank
sum test was performed for comparison of the two indepen-
dent sampling conditions. This statistical test is well suited for
the small evaluated sample size and because the VOC intensi-
ties are not normally distributed.
3 Results and discussion
3.1 Influence of everyday factors on the
breath profile
The exhaled ethanol concentration (m/z 47) increased after the
condition "eating cake" as shown in Fig. 3. This is attributed
to the function of endogenous ethanol in the carbohydrate
metabolism in the small intestine [7]. The breakdown of com-
plex carbohydrates to glucose in the lower gastrointestinal
tract is mediated by endogenous ethanol which is known to
increase the permability of epithelial and colon cells making
it available for glycolysis [7]. In contrast, the condition "eat-
ing lunch" had no significant influence on the average ethanol
abundance (Fig. 2).
Related to elevated endogenous ethanol abundance that is in-
volved in the intestinal glucose transport, we also found an in-
creased level of breath acetone (m/z 59) as a metabolic byprod-
uct (Fig. 3). We observed a slight decrease in acetone abun-
dances with high physiological variation in both male and fe-
male subjects.
cdbme_2022_8_2.pdf 262 8/29/2022 5:45:49 PM
M. Fachet et al., Breath gas variability in health volunteers
Fig. 1: Comparison of differences in inter-individual variations of
selected VOCs after getting up and before brushing teeth related
to subject gender.
Previous studies have shown that endogenous breath isoprene
is a product of the mevalonate pathway related to the biosyn-
thesis of cholesterol [7]. Under all three investigated condi-
tions, a decrease in endogenous isoprene (m/z 69) level com-
pared to the control sample was observed (Fig. 1 - 3). Methanol
(m/z 33) showed the lowest variabilities in its median and is
least sensitive to the influence of the investigated everyday life
influencing factors. In addition, we observed higher average
concentrations of VOCs for male subjects compared to female
subjects. The non-parametric Wilcoxon rank sum test shown
in Table 1 illustrates that, apart from m/z 33 (Methanol), all
other investigated breath components are significantly differ-
ent between the male and the female volunteers. In contrast,
the Wilcoxon rank sum test showed no significant differences
of the investigated volatiles before and after eating lunch.
The inter-subject variability of all measurements was com-
pared by calculating the relative standard deviation for all mea-
surements. The lowest relative standard deviation of 68 % was
observed for acetaldehyde (m/z 45). The endogenous acetone
abundance (m/z 59) had the highest variability with 115 % rel-
ative standard deviation from the mean value.
3.2 Intra- vs. inter-individual differences
of breath biomarkers
Each subject has its own “breath fingerprint”, a characteristic
profile of exhaled VOCs, which is influenced by exogenous
and endogenous factors. To check whether the inter-individual
differences between the subjects are greater than the intra-
individual differences of each individual subject, the vari-
ability between the measurements was analyzed. The results
indicated that the intra-subject differences for isopropanol
(m/z 43), acetaldehyde (m/z 45) and isoprene (m/z 69) predom-
inate, while the inter-subject differences for methanol (m/z 33),
ethanol (m/z 47) and acetone (m/z 69) were larger. Methanol
(m/z 33) and acetone (m/z 59) were found to show higher inter-
than intra-individual differences [8], which can be confirmed
by our results.
3.3 Implications for the design of clinical
studies using PTR-MS
The experimental condition "eating lunch" led to a higher vari-
ability among the study subjects due to the intake of different
meals and should therefore be avoided in clinical studies when
confounding factors should be limited to a minimum. To fur-
ther reduce the variability for ethanol (m/z 47) and acetalde-
hyde (m/z 45), food intake with a high sugar content should be
avoided prior to sampling. The experimental condition "brush-
ing teeth" had only a minor impact on the VOC variability
compared to the condition "getting up" and might not have a
significant impact on the sampling protocol in future clinical
studies (Fig. 1).
4 Summary
In summary, this work provides a tool to systematically evalu-
ate the influence of everyday influencing factors on the breath
profile of healthy subjects. This is important for future clinical
studies to limit the effect of confounding factors and to identify
a robust set of clinically relevant biomarkers for diagnostic and
therapeutic monitoring. With the simple, fast and non-invasive
technique of breath gas analysis based on PTR-MS, it gives the
opportunity to develop a possible application of breathomics
as a diagnostic and therapeutic monitoring tool.
Author Statement
Research funding: The authors state that no funding was in-
volved. Conflict of interest: Authors state no conflict of inter-
cdbme_2022_8_2.pdf 263 8/29/2022 5:45:49 PM
M. Fachet et al., Breath gas variability in health volunteers
Fig. 2: Comparison of differences in inter-individual variations of
selected VOCs before and after eating lunch related to subject
Fig. 3: Comparison of differences in inter-individual variations
of selected VOCs before and after eating cake related to subject
Tab. 1: Significance testing of breath metabolites for discrimation
between subject gender and influencing factors.
p values for
Ion Tentative female vs. before and
(m/z) compound male volunteers after lunch
33 Methanol 0.14 0.84
43 Isopropanol 0.01 0.38
45 Acetaldehyde 0.02 0.76
45 Ethanol 0.02 0.61
45 Acetone 0.03 0.68
45 Isoprene 0.03 0.44
[1] Brunner C, Szymczak W, Höllriegl V, Mörtl S, Oelmez
H, Bergner A, Huber RM, Hoeschen C, Oeh U. Discrim-
ination of cancerous and non-cancerous cell lines by
headspace-analysis with PTR-MS. Anal Bioanal Chem
[2] Duffy E, Jacobs MR, Kirby B, Morrin Aoife. Probing skin
physiology through the volatile footprint: Discriminating
volatile emissions before and after acute barrier disruption.
Experimental Dermatology 2017;10:919-925.
[3] Ehmann R, Boedeker E, Friedrich U, Sagert J, Dippon J,
Friedel G, Walles T. Canine scent detection in the diagnosis
of lung cancer: revisiting a puzzling phenomenon. Eur Respir
J. 2012 Mar;39(3):669-76.
[4] Jiang C, Dobrowolny H, Gescher DM, Meyer-Lotz G, Steiner
J, Hoeschen C, Frodl T. Volatile organic compounds from
exhaled breath in schizophrenia. World J Biol Psychiatry.
[5] Kim KH, Jahan SA, Kabir E. A review of breath analysis for
diagnosis of human health. Trends in Analytical Chemistry
[6] Schallschmidt K, Becker R, Jung C, Bremser W, Walles T,
Neudecker J, Leschber G, Frese S and Nehls I. Comparison
of volatile organic compounds from lung cancer patients and
healthy controls—challenges and limitations of an observa-
tional study. J Breath Res 2016;10(4):1–17.
[7] Sukul P, Grzegorzewski S, Broderius C, Trefz P, Mittlmeier
T, Fischer DC, Miekisch W, Schubert J. Physiological and
metabolic effects of healthy female aging on exhaled breath
biomarkers. iScience; 25. 103739.
[8] Thekedar B, Szymczak W, Höllriegl V, Hoeschen C and Oeh
U. Investigations on the variability of breath gas sampling
using PTR-MS. J Breath Res 2009;3(2):1–12.
[9] Trefz P, Schmidt S, Sukul P, Schubert J, Miekisch W, Fischer,
DC. Non-Invasive Assessment of Metabolic Adaptation in
Paediatric Patients Suffering from Type 1 Diabetes Mellitus.
Journal of Clinical Medicine 2019;8:1797.
[10] Walles T. The needle in a haystack. Eur J Cardiothorac Surg.
2016 Apr;49(4):1117-8.
[11] Wang Y, Chengyin S, Jianquan L, Haihe J, Yannan C. Pro-
ton Transfer Reaction Mass Spectrometry (PTR-MS). Mass
Spectrometry Handbook, First Edition. Edited by Mike S.
Lee. 2012 John Wiley & Sons, Inc.
cdbme_2022_8_2.pdf 264 8/29/2022 5:45:49 PM
ResearchGate has not been able to resolve any citations for this publication.
Full-text available
Healthy aging driven physio-metabolic events in female hold keys to complex in vivo mechanistic links and systemic cross-talks. Effects from basic changes at genome, proteome, metabolome and lipidome levels are often reflected at the up-stream phenome (e.g. breath volatome) cascades. Here, we have analysed exhaled volatile metabolites (measured via real-time mass-spectrometry based breathomics) data from 204 healthy females, aged between 07 – 80 years. Age related substance-specific differences were observed in breath biomarkers. Exhalation of blood-borne endogenous organosulfur, short-chain fatty acids, alcohols, aldehydes, alkene, ketones and exogenous nitriles, terpenes and aromatics have denominated interplay between endocrine differences, energy homeostasis, systemic microbial diversity, oxidative stress and lifestyle. Overall marker expressions were suppressed under daily oral contraception. Young homosexual/lesbian adults turned out as breathomic outliers. Previously proposed disease-specific breath biomarkers should be re-evaluated upon aging effects. Breathomics offers a non-invasive window towards system-wide understanding and personalized monitoring of aging i.e. translatable to gerontology.
Full-text available
An analysis of exhaled volatile organic compounds (VOC) may deliver systemic information quicker than available invasive techniques. Metabolic aberrations in pediatric type 1 diabetes (T1DM) are of high clinical importance and could be addressed via breathomics. Real-time breath analysis was combined with continuous glucose monitoring (CGM) and blood tests in children suffering from T1DM and age-matched healthy controls in a highly standardized setting. CGM and breath-resolved VOC analysis were performed every 5 minutes for 9 hours and blood was sampled at pre-defined time points. Per participant (n = 44) food intake and physical activity were identical and a total of 22 blood samples and 93 minutes of breath samples were investigated. The inter-individual variability of glucose, insulin, glucagon, leptin, and soluble leptin receptor relative to food intake differed distinctly between patients and controls. In T1DM patients, the exhaled amounts of acetone, 2-propanol, and pentanal correlated to glucose concentrations. Of note, the strength of these correlations strongly depended on the interval between food intake and breath sampling. Our data suggests that metabolic adaptation through postprandial hyperglycemia and related oxidative stress is immediately reflected in exhaled breath VOC concentrations. Clinical translations of our findings may enable point-of-care applicability of online breath analysis towards personalized medicine.
Full-text available
Patient prognosis in lung cancer largely depends on early diagnosis. The exhaled breath of patients may represent the ideal specimen for future lung cancer screening. However, the clinical applicability of current diagnostic sensor technologies based on signal pattern analysis remains incalculable due to their inability to identify a clear target. To test the robustness of the presence of a so far unknown volatile organic compound in the breath of patients with lung cancer, sniffer dogs were applied. Exhalation samples of 220 volunteers (healthy individuals, confirmed lung cancer or chronic obstructive pulmonary disease (COPD)) were presented to sniffer dogs following a rigid scientific protocol. Patient history, drug administration and clinicopathological data were analysed to identify potential bias or confounders. Lung cancer was identified with an overall sensitivity of 71% and a specificity of 93%. Lung cancer detection was independent from COPD and the presence of tobacco smoke and food odours. Logistic regression identified two drugs as potential confounders. It must be assumed that a robust and specific volatile organic compound (or pattern) is present in the breath of patients with lung cancer. Additional research efforts are required to overcome the current technical limitations of electronic sensor technologies to engineer a clinically applicable screening tool.
Objectives: This study aims to find out whether volatile organic compounds (VOCs) from exhaled breath differ significantly between patients with schizophrenia and healthy controls and whether it might be possible to create an algorithm that can predict the likelihood of suffering from schizophrenia. Methods: To test this theory, a group of patients with clinically diagnosed acute schizophrenia as well as a healthy comparison group has been investigated, which have given breath samples during awakening response right after awakening, after 30 min and after 60 min. The VOCs were measured using Proton-Transfer-Reaction Mass Spectrometry. Results: By applying bootstrap with mixed model analysis (n = 1000), we detected 10 signatures (m/z 39, 40, 59, 60, 69, 70, 74, 85, 88 and 90) showing reduced concentration in patients with schizophrenia compared to healthy controls. These could safely discriminate patients and controls and were not influenced by smoking. Logistic regression forward method achieved an area under the receiver operating characteristic curve (AUC) of 0.91 and an accuracy of 82% and a machine learning approach with bartMachine an AUC of 0.96 and an accuracy of 91%. Conclusion: Breath gas analysis is easy to apply, well tolerated and seems to be a promising candidate for further studies on diagnostic and predictive clinical utility.
Volatile organic compounds emitted by human skin were sampled before and after acute barrier disruption of the volar forearm to investigate the significance of this approach to skin physiology research. A small wearable housing integrating a solid phase micro-extraction fibre permitting rapid enclosed headspace sampling of human skin volatiles is presented, enabling non-invasive sample collection in 15 minutes, in a comfortable wearable format. Gas chromatography-mass spectrometry was utilised to separate and identify the volatile metabolites. A total of 37 compounds were identified, with aldehydes (hexanal, nonanal, decanal), acids (nonanoic, decanoic, dodecanoic, tetradecanoic and pentadecanoic acids) and hydrocarbons (squalane, squalene) predominant within the chemical profile. Acute barrier disruption was achieved through tape stripping (TS) of the stratum corneum (SC) to determine the impact on the volatile signature. Principle component analysis demonstrated there to be a discriminating volatile signature before and after TS. The dysregulation of significant features was examined. Several compounds derived from sebaceous components and their oxidation products were altered following barrier disruption, including squalane, squalene, octanal and nonanal. The up-regulation of glycine was also observed, which may indicate a perturbation to the skin's natural moisturising factor production. TS impacted the hydro-lipid film that functions within the skin barrier, resulting in a differing volatile signature from affected skin. This provides a valuable non-invasive approach for scientific and clinical studies in dermatology, particularly around dermatological disorders associated with compromised barrier function. This article is protected by copyright. All rights reserved.
This paper outlines the design and performance of an observational study on the profiles of volatile organic compounds (VOCs) in the breath of 37 lung cancer patients and 23 healthy controls of similar age. The need to quantify each VOC considered as a potential disease marker on the basis of individual calibration is elaborated, and the quality control measures required to maintain reproducibility in breath sampling and subsequent instrumental trace VOC analysis using solid phase microextraction-gas chromatography-mass spectrometry over a study period of 14 months are described. Twenty-four VOCs were quantified on the basis of their previously suggested potential as cancer markers. The concentration of aromatic compounds in the breath was increased, as expected, in smokers, while lung cancer patients displayed significantly increased levels of oxygenated VOCs such as aldehydes, 2-butanone and 1-butanol. Although sets of selected oxygenated VOCs displayed sensitivities and specificities between 80% and 90% using linear discriminant analysis (LDA) with leave-one-out cross validation, the effective selectivity of the breath VOC approach with regard to cancer detection is clearly limited. Results are discussed against the background of the literature on volatile cancer marker investigations and the prospects of linking increased VOC levels in patients' breath with approaches that employ sniffer dogs. Experience from this study and the literature suggests that the currently available methodology is not able to use breath VOCs to reliably discriminate between cancer patients and healthy controls. Observational studies often tend to note significant differences in levels of certain oxygenated VOCs, but without the resolution required for practical application. Any step towards the exploitation of differences in VOC profiles for illness detection would have to solve current restrictions set by the low and variable VOC concentrations. Further challenges are the technical complexity of studies involving breath sampling and possibly the limited capability of current analytical procedures to detect unstable marker candidates.
IntroductionHow to Identify Isomeric/Isobaric CompoundsApplicationsConclusions and ProspectAcknowledgmentsReferences
In this review, we describe technical developments in breath analysis and its applications in clinical diagnosis, monitoring disease state, and assessing environmental exposure. Breath tests have been successfully employed in clinical analyses for symptoms including lung disease, oxidative stress, gastrointestinal disease, metabolic disorders, and Helicobacter pylori infection. Although gas chromatography has been used mainly for the analysis of volatile constituents in breath samples, other techniques (e.g., sensors and lasers) have also been used satisfactorily. The analytical results of breath analysis can be derived both qualitatively and quantitatively. However, evaluation of the data from different approaches remains insufficient because of the lack of standardized procedures and poor methods of validation. Further research is therefore required to expand the applicability of breath analysis in clinical diagnosis of diseases.
Breath gas analysis is a promising technology in the frame of medical diagnostics. By identifying disease-specific biomarkers in the breath of patients, a non-invasive and easy method for early diagnosis or therapy monitoring might be developed. However, to verify this potential and develop diagnostic tools based on breath gas analysis one essential prerequisite is a low variability in measurement of exhaled volatile organic compounds. Therefore, a study has been undertaken in order to identify possible artefacts within the application of a breath gas test in practice, for which the breath gas is analysed by proton transfer reaction-mass spectrometry (PTR-MS). After validating the low instrumental variability by repeatedly measuring standard gas, the variability of breath gas sampling has been evaluated. The latter has been carried out by measuring single breath gas samples (mixed expiratory breath) collected over different periods of time such as 1 min (10 volunteers, 4 breath gas samples each), 1 h (10 volunteers, 11 breath gas samples each) and several days (11 volunteers, 10 breath gas samples each). The breath gas samples were collected in Teflon bags and consecutively measured with PTR-MS. It was found that those samples collected within 1 min and 1 h show a low variability. This was, however, not the case for samples being collected over longer periods of time (15-70 days). Under these circumstances, many volatile organic compounds (VOCs) showed significant day-to-day variation in concentration, although the breath collection had been performed under the same conditions (similar sampling time, sampling technique, sample storage time, measurement conditions, etc). This large variation might be assigned to the influence of room air VOCs, which have been investigated in this work, or with other parameters which will be discussed. It was also found that the variability in the measurement of exhaled concentrations of methanol, acetone and isoprene within different individuals (inter individual variability) is much higher than differences in the same volunteer (intra individual variability) measured over a longer time interval.