Acute Effects of Endurance Exercise With Moderate and High Intensity on Breast Milk Composition Among Women With Overweight/Obesity
Phase: NA
Conditions: Breast Milk Collection, Overweight and Obesity
Sponsor: Norwegian University of Science and Technology

Study protocol NCT05745922 
 
V. 3.0 26.02.2025 
 
1 
 
 
 
Acute Effects of Endurance Exercise with Moderate and 
High Intensity on Breast Milk Composition Among Women 
with Overweight/Obesity 
 
Acronym: YT 
 
NCT number: NCT05745922 
 
Document date: 26.02.2025 
 
 
 

Study protocol NCT05745922 
 
V. 3.0 26.02.2025 
 
2 
 
Acute effects of moderate- and high-intensity endurance exercise on breastmilk 
composition among women with overweight/obesity 
 
Background 
The period from conception to 2 years of age, often referred to as the “first 1000 days”, is considered 
the most critical for the pathophysiological disorders leading up to obesity in childhood and later life.1 
The idea that nutrition during specific windows in early life influences later health is part of a broader 
concept called programming, which reflects the impact of a stimulus or insult during a sensitive time-
period in producing long-term changes in the structure or function of the organism.2 Extensive 
evidence in animal models shows that nutrition during a brief critical window in early postnatal life 
programmes obesity, insulin resistance, dyslipidaemia, cardiovascular disease, and even lifespan.3 
Also, observational data from human cohorts confirm that faster weight gain in early infancy is 
associated with a greater risk of later obesity4-9 Indeed, rapid weight gain in the first 3 months of life 
is associated with several determinants of cardiovascular disease and type 2 diabetes in early 
adulthood.10 The key limitation, however, of such observational studies is the lack of experimental 
evidence for a causal role of early nutrition in programming human health.  
Identification of the molecular mechanisms contributing to rapid weight gain during infancy 
can guide strategies for optimal nutrition to prevent the development of childhood obesity, and 
thereby reduce the risk for type 2 diabetes and cardiovascular diseases later in life. During early 
postnatal life, the role of breastfeeding for later-life obesity is the one of the most recognised factors 
at play when discussing the nutritional background of childhood obesity. It has become clear during 
the recent decade that breastmilk composition is influenced by genetic variations and environmental 
factors, including geographical location, parity and mode of delivery, as well as lifestyle factors such 
as maternal smoking, body mass index (BMI) and diet.11-16 One behavioural factor which has received 
very little attention in this regard is physical activity/exercise.  
Regular exercise training is a formidable regulator of overall systemic metabolism through 
both acute effects driven by each exercise bout and through chronic adaptations in multiple tissues. 
Emerging work shows that the beneficial effects of exercise are not limited to adaptations within each 
tissue but from integration of inter-tissue communication by a variety of signalling molecules, 
hormones and cytokines collectively named ‘exerkines’.17,18 (Fig. 1) There are, however, little 
knowledge about how exercise affects human breastmilk.  
 
 
Fig. 1. Exercise induce multiple 
molecular adaptations in the heart, 
adipose tissue, the pancreas, skeletal 
muscle, circulation, the liver, and the 
brain, 
with 
inter-organ 
crosstalk. 
Together 
these 
molecular 
factors 
contribute to the exercise-induced 
health effects, e.g., improved insulin 
sensitivity and reduced hyperlipidemia, 
which improves cardiovascular and 
metabolic 
health. 
There 
is 
little 
evidence 
for 
exercise-induced 
adaptations 
in 
human 
breastmilk 
(beyond increased levels of single 
metabolites acutely after exercise).  
 

Study protocol NCT05745922 
 
V. 3.0 26.02.2025 
 
3 
 
Human Milk Lipids 
Human milk is rich in lipids, and one class of lipids that will be especially interesting are so-called 
“lipokines” since these lipids act as signalling molecules and can regulate inflammation and influence 
systemic metabolism.19-21 Some lipokines have been discovered in human milk. Yu and colleagues 
recently identified a breastmilk-specific lipid species (an alkylglycerol), which may protect the infant 
from obesity.22 The role of this signalling molecule is to impede the transformation of beige adipose 
tissue to lipid-storing white adipose tissue in the infant, suggesting that breastfeeding may activate 
adipocyte thermogenesis. Whether the abundance of this lipokine is influenced by maternal exercise 
is unknown. Another identified lipokine, 12,13-dyhydroxy-9Z-octadecenoic acid (12,13-diHOME) 
was shown to regulate brown adipose tissue fuel uptake and thermogenesis in both mice and 
humans.23 It was recently reported that this lipokine is present in human milk and that its abundance 
in breastmilk is inversely associated with infant adiposity24, yet again suggesting that differences in 
milk lipid composition can be functionally related to early-life obesity risk. 12,13-diHOME increases 
fatty acid uptake in skeletal muscle and its abundance in plasma has been shown to increase in 
response to acute exercise.25,26 In the work I was involved in during my research stay in Australia, 
however, we found no sustained increase in 12,13-diHOME after daily exercise training (Moholdt et 
al, in review.), which suggest that these effects are transient. Even if recent preliminary data suggest 
an increase in breastmilk 12,13-diHOME in most women after acute exercise24, there is no evidence 
for a lasting effect or, if so, whether this effect plays a causal role in early childhood obesity 
development.  
Furthermore, recent data indicate that the relative quantity of short-chain fatty acids in human 
breastmilk play a role in weight gain and adiposity during infancy, with evidence for negative 
associations between short-chain fatty acids (butyrate, formic acid, and acetate) with measures of 
infant adiposity between the ages of 3 and 12 months.27 These findings are supported by experimental 
data from animal models that identified a role for short-chain fatty acids in regulating lipid 
metabolism, insulin sensitivity, low-grade inflammation and body weight gain.28,29 Variability in fatty 
acid composition in breastmilk may partially explain inconsistent protective effects of breastfeeding 
against excessive infant adipose deposition. In line with this, exposures to different subgroups of 
short-chain fatty acids (the arachidonic acid/docosahexaenoic + eicosapentaenoic acid ratio) during 
the first 4 months of life may contribute to the way infants accumulate adipose tissue.30 The relative 
abundance of several lipid metabolites changes acutely after exercise, in a direction opposite of what 
has been implicated in cardiometabolic diseases.26 We discovered that exercise alters the relative 
abundance of metabolites involved in lipid metabolism, with a partial reversal of high-fat diet-induced 
increase in sphingolipid synthesis.31 Consequently, exercise might modify the relative abundance of 
lipid components also in breastmilk, and such exercise-induced effects can have implications for 
infant growth.  
 
Cytokines, Adipokines and Hormones 
Breastmilk contains several non-nutritive bioactive components, including hormones and cytokines. 
There is evidence that human milk exhibits substantial individual variation in the concentration of 
cytokines, adipokines and appetite-regulating hormones. Breastmilk insulin, insulin growth factor-1 
(IGF-1) and leptin levels have been reported to be higher in the milk of mothers with overweight and 
obesity32-35 and different concentrations of these hormones in breastmilk has been suggested as one 
possible reason for high growth velocity in infants of women with poor metabolic health. Breastmilk 
from obese mothers has also been shown to have elevated inflammatory markers, and a pro-

Study protocol NCT05745922 
 
V. 3.0 26.02.2025 
 
4 
 
inflammatory fatty acid profile that were found to be associated with infant growth and adiposity at 
one month of age.36,37 It is well established that chronic low-grade inflammation, indicated by 
increased pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α), contributes to 
the development and progression of metabolic disorders and non-communicable diseases such as 
insulin resistance, obesity and type 2 diabetes.38,39 Regular exercise induces suppression of TNF-α-
induced insulin resistance, and this is one of the pathways explaining the protective effects of exercise 
against chronic diseases associated with low-grade inflammation.40 To our knowledge, only one 
previous study exists on the associations between exercise and cytokines in breastmilk.41 The data 
from this observational study of 58 women showed that exercise was associated with elevated 
proinflammatory cytokines in the milk. These correlational data do, however, not provide any 
information about a possible causal relationship.  
 
Human Milk Oligosaccharides (HMOs)  
Human breastmilk contains a host of substances that may have mechanistic roles for metabolic health 
in early childhood, such as macronutrients, micronutrients, metabolic hormones, adipokines, and 
proinflammatory markers.33,42,43 In particular, human milk oligosaccharides (HMOs) have recently 
been linked to early infancy growth and body composition.14,44,45 Specifically, 2’-fucosyllactose, 
which is the most abundant HMO, was positively associated with infant weight gain between birth 
and 5 months44, whereas 6’sialyllactose and lacto-N-neotetraose have been reported to be inversely 
associated with measures of infant adiposity.44,46 These findings indicate that certain HMOs can play 
a role in infant growth.  
It was recently discovered that exercise training increases the abundance of the HMO 
3’sialyllactose in mouse milk, and that this metabolite is a critical mediator to improve metabolic 
health and cardiac function in mouse offspring.47 In the same publication, the researchers reported 
that 3’sialyllactose levels in human breastmilk two months postpartum were correlated with average 
steps taken per day. One main shortcoming in human breastmilk research is that almost all available 
data are from observational studies. As such, little is known about cause and consequences, and the 
associations reported may be solely due to confounding factors (such as dietary intake).  
 
Exercise and Human Breastmilk 
Exercise represents a major challenge to whole-body homeostasis and affects multiple cells, tissues, 
and organs in response to the increased metabolic activity of contracting skeletal muscles.17 It was 
recently shown that there is a dramatic shift in > 80% of annotated metabolites in the circulating 
metabolome in response to a single endurance-type exercise session of just 12 minutes, with 
beneficial alterations in metabolites representing key metabolic pathways central to obesity, insulin 
resistance, and inflammation.26 Such alterations can help explain the broad exercise-induced benefits 
on cardiovascular and metabolic health.  
Apart from the scarce evidence on exercise-induced adaptations in breastmilk, there is very 
little knowledge about how maternal exercise training affects milk composition. Women are 
recommended to be physically active after childbirth, but the emphasis is on maternal weight control 
and fitness. Current guidelines for exercise in lactating women are sparse. The most recent 
recommendations from the American College of Obstetricians and Gynecologists48 merely states that 
“regular aerobic exercise in lactating women has been shown to improve maternal cardiovascular 
fitness without affecting milk production, composition, or infant growth.” These recommendations 
are based on observational studies49,50 and one small RCT51 from the 1990s, before today’s 

Study protocol NCT05745922 
 
V. 3.0 26.02.2025 
 
5 
 
advancements in various ‘omics’ technologies and the application of computational and systems 
biology approaches. Furthermore, no prior studies have investigated the role of exercise intensity for 
adaptations in breastmilk composition. There is now substantial evidence for superior effects of high-
intensity exercise, as compared to moderate-intensity exercise, in a range of clinically relevant health 
outcomes.52,53  
 
Aim and hypotheses 
The overall aim of this project is to determine the acute effect of maternal exercise on breastmilk 
composition. We will also compare the effects of moderate versus high intensity exercise on 
breastmilk composition. Our hypothesis is that: Exercise will induce substantial changes in 
breastmilk composition after a single endurance exercise session, with greater effects of high-
intensity exercise than of moderate intensity exercise.  
 
Methods 
 
Participants  
We will include 20-25 women with BMI > 25 kg/m2 who are exclusively breastfeeding a singleton 
term infant. Participants will provide a written informed consent.  The assessments will be completed 
between 5 and 12 weeks postpartum, with the first assessment 5-9 weeks postpartum. No formal 
power calculation is possible for the acute study due to the exploratory nature of the research. We 
will continue including participants until we have complete data for all three sets of conditions for at 
least 20 participants. The crossover design of this study will decrease the interindividual variation in 
the statistical analysis. The women will serve as their own controls, which will remove any inter-
individual biological variance in breastmilk. 
 
Experimental design  
This will be a randomised cross-over study, in which all participants undergo three different 
conditions, in random order (Fig. 2):  
1) Moderate intensity exercise; 48 min at ~ 70% of heart rate maximum  
2) High-intensity exercise; 4 x 4 min at 90-95% of heart rate maximum 
3) No exercise. Resting in the lab. 

Study protocol NCT05745922 
 
V. 3.0 26.02.2025 
 
6 
 
 
 
 
 
 
 
 
 
Participants will walk or run on a treadmill during both exercise conditions. Inclusion criteria: 
Besteen 5+6 and 8+6 weeks postpartum at the first condition in the study, exclusively breastfeeding 
a term infant. Exclusion criteria: Known cardiovascular disease, type 1 or 2 diabetes. 
Breastmilk samples (~ 25 mL) will be collected at 4 timepoints in each condition. Samples 
obtained after exercise and in the control condition (no exercise) will be compared to a ‘baseline’ 
sample obtained at the first breastfeeding in the morning (06-0700 h) on the same day (Fig 2). Prior 
to the experimental protocol, all participants will undergo a maximal effort exercise test to establish 
their cardiorespiratory fitness (maximum oxygen uptake) and heart rate maximum. This test will be 
undertaken at a separate day, at least two days prior to the first experimental condition, and takes 
about 30 minutes. On the same day, we will also estimate the participants’ body composition using a 
bioimpedance scale (InBody 720), and infant weight and length. We will estimate infant body 
composition using biompedance (BioScan tough i8-nano).  
  
 
Standardised meals 
Participants will be asked to register their last meal the day before the first day of experiment, as well 
as the breakfast and lunch on the first day of experiment using a food diary. They will be asked to 
repeat the exact same meals at standardised time-points in the two following days of experiment (Fig. 
2).  
 
Questionnaires 
Participants will complete the International Physical Activity Questionnaire (IPAQ) at baseline. They 
will also provide information on their age, ethnicity, date of delivery, the infant’s birth weight, length 
and head circumference, and whether they are smoking or taking medications.  
 
Breastmilk sampling 
Breastmilk will be sampled by the women themselves, using an electric breast pump provided to them 
(Medela Symphony, Switzerland). Participants will be given a detailed instruction sheet and a link to 
a video for milk collection procedures. Breastmilk will be collected at the described time-points in 
the experimental protocol. For each mother, breastmilk will be collected from one breast (of her own 
Fig. 2. Exercise will take place at 1000h, and 
breastmilk will be sampled at 0 h (immediately 
after exercise), + 1 h and + 4 h after exercise/no 
exercise. Dietary intake will be standardised on 
the day before and on the day of the 
experiment. Moderate intensity exercise (A), 
High intensity exercise (B) and No exercise (C) 
will be separated by one week and will be 
undertaken in random order.  

Study protocol NCT05745922 
 
V. 3.0 26.02.2025 
 
7 
 
choice) for the entire study. The collected single full breast milk samples will be mixed and aliquoted 
into 10 x 1.8 mL sterile cryotubes and 1 x ~20 mL sterile vial, with the remaining milk for the mother 
to use. Samples will be frozen immediately after collection. Samples obtained after exercise training 
will be immediately transferred (on ice) to -80°C. The remaining samples (morning samples) will be 
frozen by the participants themselves at -20°C for up to 4 days before it will be transferred (cold 
chain) to the laboratory where it will be frozen at -80°C until analysis. The 10 x 1.8 mL aliquots will 
be used in the planned analyses, described below, whereas the additional 20 mL sample will be stored 
in a biobank for future analyses when additional funding and/or collaborations become available. 
 
Breastmilk composition  
Targeted NMR Metabolomics 
Metabolic profiling of breastmilk will be analysed using nuclear magnetic resonance (NMR) 
spectroscopy using a Bruker Avance III Ultrashielded Plus 600 MHz spectrometer (Bruker Biospin 
GmbH, Germany) at NTNU MR Core Facility. After removal of residual lipids and proteins as 
previously described54, each filtered sample (350 μL) will be mixed with 350 μL of 0.1 M phosphate 
buffer solution (pH 7.4) containing sodium 3-trimethylsilyl-(2,2,3,3-2-H3)-1-propionate (TSP) (final 
concentration 2 mM9, and then 650 μL will be transferred into a 5 mm wide NMR tube. Metabolites 
will be assigned using Bruker AMIX software v.2.5 and Chenomx v.7.11 (Chenomx Inc., Canada), 
matching spectra to reference databases of metabolites. A mother’s secretor status reflects her blood 
group antigens and determines her ability to synthesise certain HMOs, thus the secretor status for 
each individual will be determined by the high abundance (secretor) or near absence (non-secretor) 
of the HMO 2′-fucosyllactose in the respective milk samples with a cutoff of 100 nmol 2′FL/mL, as 
previously described.55 Secretor status will be accounted for in the analysis by doing sub-group 
analyses of secretors vs. non-secretors.  
 
Untargeted Global Metabolomics and Complex Lipid Panel Analysis 
In parallel, untargeted metabolomics profiling panels will be applied as an unbiased screening for the 
relative concentrations of metabolites. These analyses will be subcontracted to Metabolon, an 
internationally renowned metabolomics service company who provides high sensitivity analyses of 
more than 1000 metabolites. The principal investigator has previous experience with Metabolon data 
from a diet-exercise study.31 Samples will be analysed using ultra-high-performance liquid 
chromatography-tandem mass-spectrometry (UPLC-MS/MS; positive mode), UPLC-MS/MS 
(negative mode) and gas chromatography-MS (GC-MS).56 The Metabolon platform combines 
multiple MS methods and a laboratory information managements system with the industry’s largest 
reference library of authenticated metabolite standards and has a suite of patented informatics and 
quality-control software. These features collectively allow us to overcome the ‘signal-to-noise’ 
challenges of other metabolomics platforms. In addition to the global metabolomics profiling, we will 
undertake Complex Lipid Panel profiling at Metabolon. Complex lipids are a diverse class of 
metabolites that play important roles in the development of metabolic diseases and inflammation. 
Because these metabolites have a diverse array of chemical structures and a high degree of isomeric 
overlap, lipids are exceptionally challenging to accurately identify and quantify. The Complex Lipid 
Panel, developed by Metabolon, is currently the best (and only) lipidomic platform to provide 
complete speciation data. The Complex Lipid Penal identifies up to 1,100 individual lipid species, 
offering unparalleled insight into the lipidome.  
 

Study protocol NCT05745922 
 
V. 3.0 26.02.2025 
 
8 
 
Cytokine and hormone profiling  
We will measure a network of 27 cytokines of relevance for inflammatory status57 (Human Cytokine 
Group I multiplex panel) using Luminex xMAP Technology on a Bio-Plex 200 system (Bio-Rad) at 
the Cellular and Molecular Imaging Core Facility (CMIC), NTNU. Since subclinical mastitis, which 
is a common condition, induces substantial changes in inflammatory breastmilk components58, 
information of mastitis prevalence will be recorded and adjusted for in the statistical analyses. 
Breastmilk insulin, leptin, adiponectin will be measured by enzyme-linked immunosorbent assays 
(ELISA) using DS2 ELISA processing system (Dynex Technologies) at the Dept. of Circulation and 
Medical Imaging.  
 
Micro-RNA sequencing 
We will profile miRNA after sample preparation for extracellular vesicle enrichment of cell- and debris-free, 
defatted breastmilk, by precipitation (ExoQuick Exosome Precipitation Solution). Isolated RNA will be 
sequenced at NTNU’s internal genomics facility with their standard small RNA protocol and Illumina’s 
TruSeq SmallRNA Library Preparation Kit as per manufacturer’s instructions and sequencing with a NextSeq 
HighOutput flow cell on a NextSeq 500 instrument (Illumina Inc.). 
 
Biobanking and future directions 
We will obtain additional milk samples for later (when funding and/or new collaborations become 
available; from the Research Council Norway, Horizon 2020/Horizon Europe, or other funding 
bodies). The samples will be stored in a research biobank specific to this project, with Trine Moholdt 
as responsible for the biobank.  
 
Ethics and privacy protection 
Participation is voluntary and the participants can withdraw their written consent at any time. They 
can also request that their samples are destroyed, if they have not already been included in analyses. 
The project involves very little risk to the participants, except for a theoretical risk of an adverse event 
during exercise. Women are recommended to exercise postpartum. The knowledge gained through 
this project is highly needed and therefore outweighs the potential risk of exercise.  
Samples may be transferred to other countries. In such instances, only the biological material 
(breastmilk) and ID numbers linking the participant to the sample will be transferred, and no other 
information about the participant.  
 
Team, environment, and facilities 
This project will be undertaken at the Dept. of Circulation and Medical Imaging, NTNU. We will use 
facilities provided by NTNU, at St.Olav’s Hospital, including: the exercise physiology laboratory at 
the NextMove Core Facility, the Dept. of Circulation and Medical Imaging’s laboratories at the 3rd 
floor at AHL, laboratories at the Dept. of Clinical and Molecular Medicine, NTNU, and the MR Core 
Facility. Principal investigator is Researcher Trine Moholdt, PhD, Dept. of Circulation and Medical 
Imaging (ISB), NTNU. Collaborators: PhD student Maëliss Lemoine, ISB, NTNU, PhD student 
Emily Rose Ashby, ISB, NTNU, Postdoc Ankit Tanwar, ISB, NTNU, Reesarcher Marta Riise 
Moksnes, ISB, NTNU, Medical student Rebecca Lyng Holm, ISB, NTNU, Prof. Ann-Charlotte 
Iversen, Dept. of Clinical and Molecular Medicine (IKOM), NTNU, Staff Engineer Guro Rosvold, 
Msc, ISB, NTNU, Senior Engineer Trygve Andreassen, ISB, NTNU, Senior Engineer Liv Ryan, 
IKOM, NTNU, Senior Engineer Ragnhild Røsbjørgen, ISB, NTNU, Ass. Professor Guro 

Study protocol NCT05745922 
 
V. 3.0 26.02.2025 
 
9 
 
Giskeødegård, Dept. of Public Health and Nursing (ISM), NTNU, Ass. Professor Melanie Rae 
Simpson, ISM, NTNU, Professor Pål Sætrom, IKOM, NTNU.  
 
Budget 
The costs for the project are covered by an ERC Starting Grant to Trine Moholdt.  
 
Dissemination 
The results from the project will be published in a peer-reviewed journal, with open access. We will 
also communicate our results to the public and to health care workers seeing women in reproductive 
age (such as midwives).  
 
 
References 
1. 
Mameli C, Mazzantini S, Zuccotti GV. Nutrition in the First 1000 Days: The Origin of Childhood 
Obesity. Int J Environ Res Public Health. Aug 23 2016;13(9)doi:10.3390/ijerph13090838 
2. 
Langley-Evans SC. Developmental programming of health and disease. Proc Nutr Soc. Feb 
2006;65(1):97-105. doi:10.1079/pns2005478 
3. 
Josse M, Rigal E, Rosenblatt-Velin N, et al. Programming of Cardiovascular Dysfunction by Postnatal 
Overfeeding in Rodents. Int J Mol Sci. Dec 11 2020;21(24)doi:10.3390/ijms21249427 
4. 
Baird J, Fisher D, Lucas P, Kleijnen J, Roberts H, Law C. Being big or growing fast: systematic review 
of size and growth in infancy and later obesity. Bmj. Oct 22 2005;331(7522):929. 
doi:10.1136/bmj.38586.411273.E0 
5. 
Monteiro PO, Victora CG. Rapid growth in infancy and childhood and obesity in later life--a 
systematic review. Obes Rev. May 2005;6(2):143-54. doi:10.1111/j.1467-789X.2005.00183.x 
6. 
Zheng M, Lamb KE, Grimes C, et al. Rapid weight gain during infancy and subsequent adiposity: a 
systematic review and meta-analysis of evidence. Obesity reviews : an official journal of the International 
Association for the Study of Obesity. Mar 2018;19(3):321-332. doi:10.1111/obr.12632 
7. 
Ong KK, Loos RJ. Rapid infancy weight gain and subsequent obesity: systematic reviews and hopeful 
suggestions. Acta Paediatr. Aug 2006;95(8):904-8. doi:10.1080/08035250600719754 
8. 
Druet C, Stettler N, Sharp S, et al. Prediction of childhood obesity by infancy weight gain: an 
individual-level meta-analysis. Paediatr Perinat Epidemiol. Jan 2012;26(1):19-26. doi:10.1111/j.1365-
3016.2011.01213.x 
9. 
Weng SF, Redsell SA, Swift JA, Yang M, Glazebrook CP. Systematic review and meta-analyses of risk 
factors for childhood overweight identifiable during infancy. Arch Dis Child. Dec 2012;97(12):1019-26. 
doi:10.1136/archdischild-2012-302263 
10. 
Leunissen RW, Kerkhof GF, Stijnen T, Hokken-Koelega A. Timing and tempo of first-year rapid 
growth in relation to cardiovascular and metabolic risk profile in early adulthood. Jama. Jun 3 
2009;301(21):2234-42. doi:10.1001/jama.2009.761 
11. 
Kunz C, Meyer C, Collado MC, et al. Influence of Gestational Age, Secretor, and Lewis Blood Group 
Status on the Oligosaccharide Content of Human Milk. J Pediatr Gastroenterol Nutr. May 2017;64(5):789-
798. doi:10.1097/mpg.0000000000001402 
12. 
Gómez-Gallego C, Morales JM, Monleón D, et al. Human Breast Milk NMR Metabolomic Profile 
across Specific Geographical Locations and Its Association with the Milk Microbiota. Nutrients. Sep 21 
2018;10(10)doi:10.3390/nu10101355 
13. 
Samuel TM, Binia A, de Castro CA, et al. Impact of maternal characteristics on human milk 
oligosaccharide composition over the first 4 months of lactation in a cohort of healthy European mothers. 
Sci Rep. Aug 13 2019;9(1):11767. doi:10.1038/s41598-019-48337-4 
14. 
Isganaitis E, Venditti S, Matthews TJ, Lerin C, Demerath EW, Fields DA. Maternal obesity and the 
human milk metabolome: associations with infant body composition and postnatal weight gain. Am J Clin 
Nutr. Jul 1 2019;110(1):111-120. doi:10.1093/ajcn/nqy334 

Study protocol NCT05745922 
 
V. 3.0 26.02.2025 
 
10 
 
15. 
Bravi F, Wiens F, Decarli A, Dal Pont A, Agostoni C, Ferraroni M. Impact of maternal nutrition on 
breast-milk composition: a systematic review. Am J Clin Nutr. Sep 2016;104(3):646-62. 
doi:10.3945/ajcn.115.120881 
16. 
Hascoët JM, Chauvin M, Pierret C, et al. Impact of Maternal Nutrition and Perinatal Factors on 
Breast Milk Composition after Premature Delivery. Nutrients. Feb 10 2019;11(2)doi:10.3390/nu11020366 
17. 
Hawley JA, Hargreaves M, Joyner MJ, Zierath JR. Integrative biology of exercise. Cell. Nov 6 
2014;159(4):738-49. doi:10.1016/j.cell.2014.10.029 
18. 
Thyfault JP, Bergouignan A. Exercise and metabolic health: beyond skeletal muscle. Diabetologia. 
Aug 2020;63(8):1464-1474. doi:10.1007/s00125-020-05177-6 
19. 
Cao H, Gerhold K, Mayers JR, Wiest MM, Watkins SM, Hotamisligil GS. Identification of a lipokine, a 
lipid hormone linking adipose tissue to systemic metabolism. Cell. Sep 19 2008;134(6):933-44. 
doi:10.1016/j.cell.2008.07.048 
20. 
Yore MM, Syed I, Moraes-Vieira PM, et al. Discovery of a class of endogenous mammalian lipids 
with anti-diabetic and anti-inflammatory effects. Cell. Oct 9 2014;159(2):318-32. 
doi:10.1016/j.cell.2014.09.035 
21. 
Kuda O, Brezinova M, Rombaldova M, et al. Docosahexaenoic Acid-Derived Fatty Acid Esters of 
Hydroxy Fatty Acids (FAHFAs) With Anti-inflammatory Properties. Diabetes. Sep 2016;65(9):2580-90. 
doi:10.2337/db16-0385 
22. 
Yu H, Dilbaz S, Coßmann J, et al. Breast milk alkylglycerols sustain beige adipocytes through adipose 
tissue macrophages. J Clin Invest. May 13 2019;129(6):2485-2499. doi:10.1172/jci125646 
23. 
Lynes MD, Leiria LO, Lundh M, et al. The cold-induced lipokine 12,13-diHOME promotes fatty acid 
transport into brown adipose tissue. Nat Med. May 2017;23(5):631-637. doi:10.1038/nm.4297 
24. 
Wolfs D, Lynes MD, Tseng YH, et al. Brown fat-activating lipokine 12,13-diHOME in human milk is 
associated with infant adiposity. J Clin Endocrinol Metab. Nov 2 2020;doi:10.1210/clinem/dgaa799 
25. 
Stanford KI, Lynes MD, Takahashi H, et al. 12,13-diHOME: An Exercise-Induced Lipokine that 
Increases Skeletal Muscle Fatty Acid Uptake. Cell Metab. Jun 5 2018;27(6):1357. 
doi:10.1016/j.cmet.2018.04.023 
26. 
Nayor M, Shah RV, Miller PE, et al. Metabolic Architecture of Acute Exercise Response in Middle-
Aged Adults in the Community. Circulation. Nov 17 2020;142(20):1905-1924. 
doi:10.1161/circulationaha.120.050281 
27. 
Prentice PM, Schoemaker MH, Vervoort J, et al. Human Milk Short-Chain Fatty Acid Composition is 
Associated with Adiposity Outcomes in Infants. J Nutr. May 1 2019;149(5):716-722. doi:10.1093/jn/nxy320 
28. 
Gao Z, Yin J, Zhang J, et al. Butyrate improves insulin sensitivity and increases energy expenditure in 
mice. Diabetes. Jul 2009;58(7):1509-17. doi:10.2337/db08-1637 
29. 
Li Z, Yi CX, Katiraei S, et al. Butyrate reduces appetite and activates brown adipose tissue via the 
gut-brain neural circuit. Gut. Jul 2018;67(7):1269-1279. doi:10.1136/gutjnl-2017-314050 
30. 
Rudolph MC, Young BE, Lemas DJ, et al. Early infant adipose deposition is positively associated with 
the n-6 to n-3 fatty acid ratio in human milk independent of maternal BMI. Int J Obes (Lond). Apr 
2017;41(4):510-517. doi:10.1038/ijo.2016.211 
31. 
Moholdt T, Parr EB, Devlin BL, Debik J, Giskeødegård G, Hawley JA. The effect of morning vs evening 
exercise training on glycaemic control and serum metabolites in overweight/obese men: a randomised trial. 
Diabetologia. May 19 2021;doi:10.1007/s00125-021-05477-5 
32. 
Young BE, Patinkin Z, Palmer C, et al. Human milk insulin is related to maternal plasma insulin and 
BMI: but other components of human milk do not differ by BMI. Eur J Clin Nutr. Sep 2017;71(9):1094-1100. 
doi:10.1038/ejcn.2017.75 
33. 
Fields DA, George B, Williams M, et al. Associations between human breast milk hormones and 
adipocytokines and infant growth and body composition in the first 6 months of life. Pediatr Obes. Aug 
2017;12 Suppl 1(Suppl 1):78-85. doi:10.1111/ijpo.12182 
34. 
Sims CR, Lipsmeyer ME, Turner DE, Andres A. Human milk composition differs by maternal BMI in 
the first 9 months postpartum. Am J Clin Nutr. Sep 1 2020;112(3):548-557. doi:10.1093/ajcn/nqaa098 
35. 
Kon IY, Shilina NM, Gmoshinskaya MV, Ivanushkina TA. The study of breast milk IGF-1, leptin, 
ghrelin and adiponectin levels as possible reasons of high weight gain in breast-fed infants. Ann Nutr 
Metab. 2014;65(4):317-23. doi:10.1159/000367998 

Study protocol NCT05745922 
 
V. 3.0 26.02.2025 
 
11 
 
36. 
Panagos PG, Vishwanathan R, Penfield-Cyr A, et al. Breastmilk from obese mothers has pro-
inflammatory properties and decreased neuroprotective factors. J Perinatol. Apr 2016;36(4):284-90. 
doi:10.1038/jp.2015.199 
37. 
Fields DA, Demerath EW. Relationship of insulin, glucose, leptin, IL-6 and TNF-α in human breast 
milk with infant growth and body composition. Pediatr Obes. Aug 2012;7(4):304-12. doi:10.1111/j.2047-
6310.2012.00059.x 
38. 
Ouchi N, Parker JL, Lugus JJ, Walsh K. Adipokines in inflammation and metabolic disease. Nat Rev 
Immunol. Feb 2011;11(2):85-97. doi:10.1038/nri2921 
39. 
Saltiel AR, Olefsky JM. Inflammatory mechanisms linking obesity and metabolic disease. J Clin 
Invest. Jan 3 2017;127(1):1-4. doi:10.1172/jci92035 
40. 
Petersen AM, Pedersen BK. The anti-inflammatory effect of exercise. J Appl Physiol (1985). Apr 
2005;98(4):1154-62. doi:10.1152/japplphysiol.00164.2004 
41. 
Groër MW, Shelton MM. Exercise is associated with elevated proinflammatory cytokines in human 
milk. J Obstet Gynecol Neonatal Nurs. Jan-Feb 2009;38(1):35-41. doi:10.1111/j.1552-6909.2008.00303.x 
42. 
de la Garza Puentes A, Martí Alemany A, Chisaguano AM, et al. The Effect of Maternal Obesity on 
Breast Milk Fatty Acids and Its Association with Infant Growth and Cognition-The PREOBE Follow-Up. 
Nutrients. Sep 9 2019;11(9)doi:10.3390/nu11092154 
43. 
Galante L, Pundir S, Lagström H, et al. Growth Factor Concentrations in Human Milk Are Associated 
With Infant Weight and BMI From Birth to 5 Years. Front Nutr. 2020;7:110. doi:10.3389/fnut.2020.00110 
44. 
Larsson MW, Lind MV, Laursen RP, et al. Human Milk Oligosaccharide Composition Is Associated 
With Excessive Weight Gain During Exclusive Breastfeeding-An Explorative Study. Front Pediatr. 2019;7:297. 
doi:10.3389/fped.2019.00297 
45. 
Lagström H, Rautava S, Ollila H, et al. Associations between human milk oligosaccharides and 
growth in infancy and early childhood. Am J Clin Nutr. Apr 1 2020;111(4):769-778. 
doi:10.1093/ajcn/nqaa010 
46. 
Alderete TL, Autran C, Brekke BE, et al. Associations between human milk oligosaccharides and 
infant body composition in the first 6 mo of life. Am J Clin Nutr. Dec 2015;102(6):1381-8. 
doi:10.3945/ajcn.115.115451 
47. 
Harris JE, Pinckard KM, Wright KR, et al. Exercise-induced 3'-sialyllactose in breast milk is a critical 
mediator to improve metabolic health and cardiac function in mouse offspring. Nat Metab. Aug 
2020;2(8):678-687. doi:10.1038/s42255-020-0223-8 
48. 
Physical Activity and Exercise During Pregnancy and the Postpartum Period: ACOG Committee 
Opinion, Number 804. Obstetrics and gynecology. Apr 2020;135(4):e178-e188. 
doi:10.1097/aog.0000000000003772 
49. 
Lovelady CA, Lonnerdal B, Dewey KG. Lactation performance of exercising women. Am J Clin Nutr. 
Jul 1990;52(1):103-9. doi:10.1093/ajcn/52.1.103 
50. 
Fly AD, Uhlin KL, Wallace JP. Major mineral concentrations in human milk do not change after 
maximal exercise testing. Am J Clin Nutr. Aug 1998;68(2):345-9. doi:10.1093/ajcn/68.2.345 
51. 
Dewey KG, Lovelady CA, Nommsen-Rivers LA, McCrory MA, Lönnerdal B. A randomized study of the 
effects of aerobic exercise by lactating women on breast-milk volume and composition. N Engl J Med. Feb 
17 1994;330(7):449-53. doi:10.1056/nejm199402173300701 
52. 
Jelleyman C, Yates T, O'Donovan G, et al. The effects of high-intensity interval training on glucose 
regulation and insulin resistance: a meta-analysis. Obes Rev. Nov 2015;16(11):942-61. 
doi:10.1111/obr.12317 
53. 
Ramos JS, Dalleck LC, Tjonna AE, Beetham KS, Coombes JS. The Impact of High-Intensity Interval 
Training Versus Moderate-Intensity Continuous Training on Vascular Function: a Systematic Review and 
Meta-Analysis. Sports Med. Mar 15 2015;doi:10.1007/s40279-015-0321-z 
54. 
Dessì A, Briana D, Corbu S, et al. Metabolomics of Breast Milk: The Importance of Phenotypes. 
Metabolites. Nov 20 2018;8(4)doi:10.3390/metabo8040079 
55. 
Seferovic MD, Mohammad M, Pace RM, et al. Maternal diet alters human milk oligosaccharide 
composition with implications for the milk metagenome. Sci Rep. Dec 16 2020;10(1):22092. 
doi:10.1038/s41598-020-79022-6 

Study protocol NCT05745922 
 
V. 3.0 26.02.2025 
 
12 
 
56. 
Evans AM, DeHaven CD, Barrett T, Mitchell M, Milgram E. Integrated, nontargeted ultrahigh 
performance liquid chromatography/electrospray ionization tandem mass spectrometry platform for the 
identification and relative quantification of the small-molecule complement of biological systems. 
Analytical chemistry. Aug 15 2009;81(16):6656-67. doi:10.1021/ac901536h 
57. 
Tangeras LH, Austdal M, Skrastad RB, et al. Distinct First Trimester Cytokine Profiles for Gestational 
Hypertension and Preeclampsia. Arteriosclerosis, thrombosis, and vascular biology. Nov 2015;35(11):2478-
85. doi:10.1161/atvbaha.115.305817 
58. 
Tuaillon E, Viljoen J, Dujols P, et al. Subclinical mastitis occurs frequently in association with 
dramatic changes in inflammatory/anti-inflammatory breast milk components. Pediatr Res. Apr 
2017;81(4):556-564. doi:10.1038/pr.2016.220 
+|+