Metabolic Flexibility in Patients With Early Triple-negative Breast Cancer and the Possible Effect of a Physical Exercise Intervention
Phase: NA
Conditions: Triple-Negative Breast Cancer (TNBC)
Sponsor: Universidad Europea de Madrid

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
Version 2. 17-11-2025.                                                                                                                                                                     PI: Lidia B. Alejo 
 
STUDY PROTOCOL “METABOLIC FLEXIBILITY IN PATIENTS WITH EARLY TRIPLE-NEGATIVE BREAST 
CANCER AND THE POSSIBLE EFFECT OF A PHYSICAL EXERCISE INTERVENTION” 
PI: Lidia B. Alejo 
INDEX 
1. 
INTRODUCTION ................................................................................................................ 2 
1.1 CANCER INCIDENCE ......................................................................................................... 2 
1.2 BREAST CANCER SUBTYPES .............................................................................................. 2 
1.3 GENERAL BENEFITS OF PHYSICAL EXERCISE...................................................................... 2 
1.4 DEFINITION AND RISK FACTORS OF METABOLIC FLEXIBILITY ............................................. 3 
1.5 METABOLIC INFLEXIBILITY IN THE ONCOLOGY POPULATION ............................................. 4 
1.6 PHYSICAL EXERCISE AS A TOOL FOR IMPROVING METABOLIC FLEXIBILITY ........................ 5 
2. 
OBJECTIVES ...................................................................................................................... 7 
2.1 MAIN OBJECTIVES ........................................................................................................... 7 
2.2 SECONDARY OBJECTIVES ................................................................................................. 8 
3. 
MATERIALS AND METHODS .............................................................................................. 8 
3.1 STUDY DESIGN ................................................................................................................. 8 
3.2 SAMPLE SELECTION AND PARTICIPANTS ........................................................................... 9 
3.3 MEASURING INSTRUMENTS........................................................................................... 10 
3.4 ANALYSED VARIABLES .................................................................................................... 12 
3.5 STUDY PROCEDURE ....................................................................................................... 13 
 
EXERCISE PROGRAM ................................................................................................... 16 
 
ETHICAL AND CONFIDENTIALITY ISSUES ...................................................................... 18 
 
STUDY RESEARCH TEAM ............................................................................................. 20 
 
PATIENT TIMELINE ...................................................................................................... 21 
 
STUDY BUDGET .......................................................................................................... 22 
3.6 STATISTICAL ANALYSIS .................................................................................................... 23 
REFERENCES ........................................................................................................................... 25 
APPENDICES ............................................................................................................................ 30 
APPENDIX 1 ......................................................................................................................... 30 
 
 
 
 

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
Version 2. 17-11-2025.                                                                                                                                                                     PI: Lidia B. Alejo 
 
1. INTRODUCTION 
1.1 CANCER INCIDENCE 
Cancer is considered like a main worlwide issue. It was estimated about approximately 19,9 
million of new cancer diagnoses in 2022 in the world and those will increase up to 28,00 million 
in the next two decades (1). Specifically, breast cancer (BC) represents the highest incidence 
worlwide, with approximately 2,3 million of new cases diagnosed in 2022 (1). According to 
Sociedad Española de Oncología Médica (SEOM) it was estimated about 37.682 new BC cases 
diagnoses in 2025 in Spain (1).  
There is a higher BC incidence in developed countries that could be explained due to the higher 
obesity rates, alcohol and tobacco consumption, early onset of puberty, contraceptive use and 
hormone therapies, low levels of physical activity, and giving birth at an older age (2,3). 
Furthemore to everything mentioned above, hereditary factors and age are additional risk 
factors to cancer development (2,3). Last but not least important, the existence of relatives with 
BC and/or ovarian cancer who are carriers of mutations in the BRCA1 and BRCA2 genes, among 
others, which increase the likelihood of tumour proliferation, and being over 40 years of age, 
increment the likelihood of BC diagnosis too (2,3).  
 
1.2 BREAST CANCER SUBTYPES 
Currently, diferent types of BC are categorized based on molecular subtype (64,65). On the one 
hand, the luminal A molecular subtype is characterised by expressing oestrogen receptors (ER) 
and/or progesterone receptors (PR), absence of overexpression of human epidermal growth 
factor (HER2), and a Ki-67 cell proliferation index <20% (64,65). On the other hand, the luminal 
B molecular subtype has a inmunostoquimic profile compound by positive RE and/or RP, negative 
or positive HER2 and a Ki-67 ≥20% (64,65). Finally, the triple negative molecular subtype is 
defined as one that does not express ER, PR, and HER2 (64,65).  
Specifically this last molecular subtype accounts for 10-20% of invasive BC, and they are 
biologically and clinically more agressive than other BC subtypes (64,65). Moreover, this subtype 
has a poorer prognosis and more limited treatment options than other BC subtypes (64,65).  
In this line, in a study conducted in North Carolina on patients with triple-negative breast cancer 
(TNBC) a strong association was found between triple-negative tumor diagnoses in women and 
multiple births, early menarche, low breastfeeding rates, a high waist-to-hip ratio, and high 
adiposity (66). In contrast to other tumors, it appears to be the only molecular subtype in which 
increasing age is not a risk factor; rather, a high incidence of TNBC has been observed between 
the ages of 20 and 39 (66).   
 
1.3 GENERAL BENEFITS OF PHYSICAL EXERCISE 
It has been proven that exercise has beneficial effects on the immune system that could protect 
against the development of cancer (4,5). When physical exercise is performed, exerkines are 
detach, molecules that are released into the bloodstream by different organs and tissues (4). 

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
Version 2. 17-11-2025.                                                                                                                                                                     PI: Lidia B. Alejo 
 
There are two main types of exerkines: myokines, generated by muscle fibre contraction, and 
adipokines, secreted by adipose tissue (4). 
With regard to these exerkines, specifically IL-6, a myokine released by muscle contraction, has 
an anti-tumor effect due to the way it is secreted in acute doses (6). However, the continuous 
secretion of IL-6 by other tissues has inflammatory effects and is considered pro-tumor as it 
increases the likelihood of tumor cell appearance and/or proliferation (6). Although these 
immunological benefits are not the target of study, the presence and release of these molecules 
through physical exercise directly influences the anti-inflammatory phenotype of the body's cells 
(6). It has been shown that cells with a high inflammatory profile may have mitochondrial 
dysfunction, greater oxidative stress, and a higher presence of pro-inflammatory cytokines. This 
could, in turn, alter the pathways of energy generation from the mitochondria (6).  
 
Along the same lines, different studies show how physical exercise could change the cellular 
phenotype in different tissues of the body (13,14). Thus, a decrease in certain markers of 
inflammation such as IL-6, IL-1, TNF-α, M1 macrophages, etc. has been observed with different 
exercise programs, which would shift cells toward a more anti-inflammatory phenotype (13,14). 
Low-grade chronic inflammation is a risk factor for tumor proliferation (13,14). Therefore, 
exercise could interfere with tumor proliferation processes.  
On the other hand, numerous authors reflect on the importance and potential effect that 
physical exercise can have on the metabolism of the body's cells. Various studies have been 
carried out that include concurrent training interventions in humans and evaluate certain 
metabolic parameters (15,16). The authors reported that physical exercise significantly 
influences glucose levels, insulin resistance, growth factors, fat oxidation ratio, and lactate, 
among others (15,16). Alteration of any of these markers could activate tumor signaling 
pathways, thus, posing a risk factor for tumor proliferation (15,16). Therefore, it could be 
concluded that modulation of metabolism through physical exercise plays a fundamental role in 
the development of cancer (5,15,16).  
 
1.4 DEFINITION AND RISK FACTORS OF METABOLIC FLEXIBILITY 
Metabolic flexibility is described as the body's ability to adapt to energy demands in different 
contexts (18,20). The availability of energy substrates in response to different activity 
requirements enables cell survival and homeostasis (18,20). Mitochondria, the main organelle 
in the cell, enable the oxidation of energy substrates for energy production in order to respond 
to the required intensity (20).  
The main energy substrates used by muscle to generate energy are intramuscular glycogen, 
blood glucose, triglycerides (TG), and fatty acids (FA), which come mainly from abdominal white 
adipose tissue (WAT) (18). All of these provide the necessary energy supply for muscle fibre to 
generate muscle contraction (18).  
In terms of rest, under normal health conditions, FAs and carbohydrates (CHO) are transported 
into the mitochondria to be oxidated into acetyl-CoA and enter the Krebs cycle (TCA) with its 

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
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corresponding process of obtaining adenosine triphosphate (ATP) through oxidative 
phosphorylation (OXPHOS) (20). Any alteration in the energy production process in the 
mitochondria could alters cellular homeostasis and the health of the individual (20).  
The concept of metabolic inflexibility in muscle fibre refers to the inability of mitochondria to 
clear lactate, the low capacity to oxidate lipids, and the rapid transition from fat oxidation to CHO 
(21). Along the same lines, high metabolic function will mean lower lactate levels and a higher 
FA oxidation ratio at the same intensity. Thus, people with low metabolic function have a higher 
respiratory exchange ratio (RER) both at rest and during exercise, mainly due to the rapid 
transition from fat oxidation to CHO and/or mitochondrial dysfunction in fat oxidation (21).  
Currently, many people suffer from metabolic inflexibility mainly due to unhealthy habits such 
as a diet high in processed foods, the consumption of toxins such as alcohol and drugs, regular 
use of medication for illnesses, inadequate rest and high levels of stress, and physical inactivity 
(19,20). This has a negative impact on a person's mitochondrial function and is associated with 
a high probability of developing type 2 diabetes (T2D), cardiovascular disease (CVD), metabolic 
syndrome (MS), cancer, and Alzheimer's disease (19,20). Other factors that could lead to 
mitochondrial dysfunction include age, infections, and DNA mutations (20).  
Similarly, systemic mitochondrial dysfunction has a direct impact on skeletal muscle, which 
oxidates high levels of glucose and a minimal amount of FA (18,20). Some authors also indicate 
that defects in the oxidation of these alter energy production in the mitochondria, leading to an 
accumulation of intramyocellular lipids (18,20).  
In this context, there is a new tool called near-infrared spectroscopy (NIRS), which allows the 
analysis of oxygenated haemoglobin (HbO2), deoxygenated haemoglobin (HHb) and the Tissue 
Saturation Index (TSI) (45,46). This measuring instrument has already been used to evaluate 
muscle mitochondrial capacity in numerous research (44,45,46,47,58). It therefore provides 
direct information on mitochondrial function, which indirectly determines an individual's 
metabolic flexibility. 
 
1.5 METABOLIC INFLEXIBILITY IN THE ONCOLOGY POPULATION 
On the one hand, some authors argue that in the presence of cancer there is generalized 
mitochondrial dysfunction in different tissues of the body, marked by the pathophysiology of the 
disease and by the toxicity induced by cancer treatments in these patients (11). Similarly, a 
reduction in PGC-1α levels has been observed in patients who received neoadjuvant (NA) 
chemotherapy (CTx), considered one of the coactivator proteins that control mitochondrial 
biogenesis. This would lead to an inability to produce and meet energy demands and, once again, 
to muscle dysfunction associated with both loss of function and muscle mass in cancer patients 
(7,11).  
On the other hand, it should be noted that women with BC who have been under the effects of 
CTx are more likely to gain fat mass than women of the same age without BC (7,9,10,11,22). Body 
fat gain is a side effect that occurs with cancer treatments, but it is more pronounced in the 
context of adjuvant treatment and endocrine therapies in BC (7,9,10,11,22). Excess fatty tissue 
is associated with the presence of metabolic diseases and pro-inflammatory cytokines, which 

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
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again imply metabolic and mitochondrial dysfunction. Likewise, increased fat mass has been 
linked to an increased likelihood of recurrence, disease progression, and even mortality in 
studies of patients with BC (7,9,10,11,22).  
In patients with any type of cancer, sarcopenia is associated with lower success rates of 
antineoplastic treatments, greater CTx-induced toxicity, and rapid tumor progression (7,9,11). 
Furthermore, taking into account the previous paragraph, the aforementioned predisposing 
factors could alter the body composition of cancer patients. Thus, sarcopenic obesity, 
characterized by an increase in fat mass and a decrease in muscle mass, is quite common in BC 
(7,9,11).  
The changes in weight and body composition in patients with BC are not only induced by CTx 
and the disease itself, but could also be associated with physical inactivity, a decrease in resting 
metabolic rate (TMB), excessive food intake, and/or hormonal changes (9). These changes can 
persist for years after treatments (9).  
Another factor to consider in cancer patients is the phase angle (PhA). This is defined as the 
correlation between the resistance (R) of the electrolytes contained in total body water and the 
reactance (Xc) present in cell membranes (42). Cancer treatments are known to decrease PhA as 
they may affect cell membrane integrity, causing inflammation and, in some cases, cancer-
associated cachexia (42). PhA is considered a prognostic indicator of survival and quality of life 
in cancer survivors in the clinical setting (42)Thus, low PhA values indicate low Xc and high R, or, 
in other words, poor cell membrane integrity (42). High PhA values result in high Xc and low R, 
indicating that cell membranes remain intact and that there are high levels of electrolytes and 
total body water (42).  
Therefore, PhA is considered an indirect marker of a person's metabolic flexibility due to its direct 
relationship with membrane integrity, which involves the cell itself and could lead to 
mitochondrial dysfunction (42).  
In summary, it could be concluded that the metabolic flexibility of muscle fibre in patients with 
TNBC could be reduced, mainly in patients undergoing systemic treatment, with possible 
complications in adapting to different intensities and energy demands in different everyday 
contexts. The decrease in muscle metabolic flexibility would also imply a decrease in the 
patient's strength levels and physical function, significantly impairing their quality of life.  
 
1.6 PHYSICAL EXERCISE AS A TOOL FOR IMPROVING METABOLIC FLEXIBILITY 
When faced with an increase in glycolytic flux, high-intensity exercise, or high CHO intake, the 
pyruvate molecule is not reduced to acetyl-CoA, but rather prefers to be reduced to lactate 
outside the mitochondria in order to obtain energy more quickly (18,20). High-intensity exercise 
is known to increase glycolysis, thereby increasing glucose oxidation (20). It has also been 
observed that the only mechanism that allows glucose to enter muscle fibre and other tissues, 
regardless of the presence of insulin, is muscle contraction, via the GLUT 4 transporter (18,20). 
Whether or not glucose enters the mitochondria will depend on the energy requirements of the 
activity for the individual and their metabolic flexibility (18,20).  

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
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As the intensity decreases and the duration of exercise increases, FA make a significant 
contribution to energy production, as they provide large amounts of energy more slowly (18).  
Physical exercise is key to activating the AMPK signalling pathway, an enzyme that is released in 
response to high energy requirements. When this mechanism of action is initiated, GLUT 4 
translocation occurs, and glycolysis, FA oxidation and mitochondrial biogenesis increase (with an 
increase in the coactivator protein that controls mitochondrial biogenesis, PGC-1α) (23,25). It 
has been observed that extensive continuous exercise activates AMPK hours after activity and 
that there are greater benefits as the duration of activity increases (23,24). Similarly, high-
intensity cardiovascular exercise programmes also appear to increase AMPK hours after activity 
(23,24). In addition, high-intensity sprint training has been found to increase PGC-1α expression, 
and this increase appears to be greater than with other types of training. The benefits of AMPK 
activation from both types of cardiovascular training appear to increase when the person 
performs the activity while fasting (23,24).  
In relation to the above, physical exercise is considered a primary tool for improving metabolic 
flexibility in muscle fibre and WAT (18,19,21). When AMPK and PGC-1α increases with training, 
increases in mitochondrial content and function in muscle fibre have been observed 
(18,19,21,25). This has a positive effect on the ability to oxidise FA in the mitochondria, improving 
insulin sensitivity (18,19,21). All of this would improve the ability of muscle fibre, now with a 
greater number of mitochondria, to oxidise different substrates and thus improve metabolic 
flexibility (18,19,21,26).  
Numerous studies reflect the high muscle oxidative capacity of trained endurance athletes, 
which translates into a preference for FA oxidation, preserving existing glycogen (21,26). These 
adaptations to prolonged training make them functionally more efficient in energy expenditure 
and production, with improved mitochondrial health (21,26). Athletes are able to maintain 
activity for longer and with less fatigue than the normal untrained population due mainly to the 
high mitochondrial function and density of muscle fibres (21,26).  
Another study conducted on professional cyclists who underwent an incremental exercise test 
on a cycle ergometer showed that they had a greater capacity for fat oxidation and delayed 
lactate accumulation due to the high capacity of the mitochondria to continue clearing the 
substrate, compared to the healthy active population and those with MS (21). Different authors 
have observed a practically perfect inverse correlation between fat oxidation and lactate 
concentration, and therefore this relationship has been considered an indirect marker of 
mitochondrial function and metabolic flexibility (20,21).  
There are different physiological markers that determine a person's cardiorespiratory fitness in 
an incremental exercise test on a cycle ergometer: maximum oxygen consumption (VO2máx.), 
aerobic threshold or 1 (VT1), anaerobic threshold or 2 (VT2), maximum power (Pmax), CHO 
oxidation (CHOox), and fat oxidation (FATox), among others (21).   
An improvement in these markers indicates a greater capacity to oxidise FA before switching to 
the main glycolytic pathway, and greater lactate clearance (15,21,27). Thus, professional cyclists 
have higher VO2máx.values than the normal active population, as well as a higher number of watts 
(W) generated during the incremental test (15,21,27). The results obtained in the CHOox test are 
similar, with higher values in the healthy active population and populations with metabolic 

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
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dysfunctions compared to professional cyclists, and with higher values in the FATox in professional 
cyclists throughout the test. These results once again reflect the high oxidative capacity of 
professional athletes (15,21,27). 
Scientific evidence shows the importance of physical exercise programmes for improving body 
composition at all stages of the disease in cancer patients (22,28). Specifically, reducing or 
maintaining the percentage of fat mass and maintaining or increasing muscle mass gains 
importance in the different interventions proposed in the research (22,28,29). Improved body 
composition is directly associated with lower CTx-induced toxicity and a higher probability of 
completing treatment, which could lead to higher survival rates and lower disease progression 
rates (22,28).  
Currently, there are few studies analysing the relationship between metabolic dysfunction and 
the different molecular subtypes in patients with BC (67). However, in a study conducted in 
Nigeria with 296 women diagnosed with BC and 259 healthy control women, a strong association 
was found between MS and triple-negative molecular subtype diagnoses (67). Therefore, it could 
be assumed that any alteration in metabolic markers such as hyperinsulinemia or 
hyperglycaemia could increase the risk of developing TNBC.  
Along the same lines, new research shows that increasing muscle strength and preventing fat 
mass gain is essential to good metabolic health and adequate response to treatment, as it has 
been associated with improved metabolic markers (insulin sensitivity, glycaemic control, acute 
anti-inflammatory response, etc.) (29,31). The benefits of physical exercise on mitochondrial and 
metabolic health have been shown to be independent of weight loss associated with fat loss 
(29,31).  
In summary, cancer patients, specifically those with TNBC, may suffer from systemic metabolic 
dysfunction marked by the pathophysiology of the disease, the toxicity of cancer treatments, 
and unhealthy habits (4,5,7,8,10,11,12,17,18,19,20,27). However, to date, no studies have 
described the metabolic response to exercise in this type of patient.  
Therefore, the aim is to describe the metabolic flexibility presented in participating patients with 
TNBC in the different phases of early disease. In this way, the energy substrate could be verified 
indirectly with a greater preference for muscle fibre in the different phases of early disease, and 
determine the intensity at which FA oxidation could be most effective in improving the metabolic 
profile in these patients. 
Similarly, the second aim is to verify the effects of two cardiovascular training interventions, on 
the one hand, and a strength intervention, on the other, on the metabolic profile in patients with 
TNBC in the early stages of the disease. 
 
2. OBJECTIVES 
2.1 MAIN OBJECTIVES 
- 
Describe the metabolic function of women diagnosed with TNBC at different stages of 
early-onset disease. 

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
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- 
Assess the effects of different training interventions on the metabolic function of women 
diagnosed with early-onset TNBC. 
 
2.2 SECONDARY OBJECTIVES 
- 
Analyse whether the heterogeneity of treatments in women newly diagnosed with TNBC 
modifies metabolic function in the different phases of early-stage disease. 
- 
Analyse whether the accumulation of cancer treatments and the toxicity induced by 
them modifies metabolic function in patients diagnosed with TNBC in early-stage 
disease. 
- 
Analyse the possible relationship between patient-reported outcomes (PROs) in patients 
diagnosed with early-stage TNBC and metabolic flexibility. 
- 
Compare whether possible changes in metabolic flexibility with different exercise 
interventions are associated with improvements in PROs in patients diagnosed with 
early-stage TNBC. 
- 
Check the most effective exercise intervention on the different parameters of metabolic 
flexibility in women with TNBC.   
 
3. MATERIALS AND METHODS 
3.1 STUDY DESIGN 
This is a pilot study, initially descriptive and longitudinal, followed by a randomized, open-label 
experimental phase conducted in patients with early-onset TNBC. It will involve a descriptive 
observational analysis in the first instance, followed by an experimental analysis with four groups 
using pre-post testing, including a control group (CG).  
The recruited patients will begin the descriptive part of the study upon diagnosis and, once they 
have recovered from surgery for the condition, they will begin the experimental part.  
Therefore, there will be a group consisting solely of newly diagnosed TNBC patients (D1) with 
the inclusion criteria specified in the following section. This group will only receive the general 
physical exercise recommendations of the World Health Organization (WHO) throughout 
neoadjuvant treatment and prior to breast surgery. 
In the experimental part, the initial D1 group of the sample will be divided into subgroups to 
carry out the physical exercise intervention protocol. Thus, the CG will receive the WHO's general 
physical exercise recommendations; experimental group 1 (E1) will complete 12 weeks of 
cardiovascular training around maximum fat oxidation (FATmáx) twice a week; experimental group 
2 (E2) will perform 12 weeks of cardiovascular training at maximum aerobic power (MAP) twice 
a week; and finally, experimental group 3 (E3) will complete 12 weeks of strength training twice 
a week. The four groups in the experimental design will be composed of patients with TNBC who 
have received neoadjuvant treatment and undergone surgery for their BC (Table 1). 
Table 1. Summary of groups and interventions. 

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
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STUDY PHASE 
 
DESCRIPTIVE 
 
EXPERIMENTAL 
GROUP 
D1 
D1 CG 
D1 E1 
D1E2 
D1E3 
 
 
INTERVENTION 
Metabolic 
flexibility 
description during 
neoadyuvant 
treatment 
WHO 
recommendatio
ns 
12 weeks of 
cardiovascular 
training around 
FATmáx 
12 weeks of 
cardiovascular 
training at MAP 
12 weeks of 
strength 
training 
 
3.2 SAMPLE SELECTION AND PARTICIPANTS 
The sample will consist of 40 patients with TNBC selected for convenience from the Severo 
Ochoa University Hospital (Av. de Orellana, s/n, 28914 Leganés, Madrid), the Infanta Leonor 
University Hospital (Av. Gran Vía del Este, 80, Vallecas, 28031 Madrid), and the la Princesa 
University Hospital (Calle de Diego de León, 62, Salamanca, 28006 Madrid), belonging to the 
Community of Madrid. The sample size is small due to the low number of diagnoses in this 
molecular subtype of TNBC and the estimated number of patients diagnosed each year at the 
aforementioned hospitals. Therefore, the study has limited power, as the results obtained cannot 
be extrapolated to the general oncology population or to breast cancer patients. For sample 
selection, a direct referral methodology will be used from the oncologists at the aforementioned 
hospitals to all patients with TNBC who wish to participate in the study and are seen at a medical 
consultation at the time of recruitment. Recruitment will last for one year, taking into account 
the diagnosis rates reported by the described hospitals. 
The inclusion and exclusion criteria that will determine the sample selection for the study are: 
Inclusion criteria: 
- 
Female between 20-55 years of age. 
- 
Confirmed histological new diagnosis of stage I to III triple-negative breast cancer and a 
candidate to receive systemic neoadjuvant treatment with chemotherapy +/- 
immunotherapy. 
- 
Must not have started systemic treatment for the neoplastic disease. 
- 
Participating in any external physical exercise program or sports activities during the 
experimental study phase. 
- 
Inability to understand and provide written Informed Consent (IC) (Appendix 1). 
Exclusion Criteria:  
- 
Having neurological or orthopedic disease at the time of recruitment or during the study 
evaluation and intervention.  
- 
Inability to understand Spanish language. 
- 
Presenting absolute contraindications for performing a cardiopulmonary exercise test 
(CPET) such as heart failure, myocarditis, acute pericarditis, severe aortic stenosis, aortic 
dissection, vascular toxicity, uncontrolled severe arterial hypertension, uncontrolled 
severe cardiac arrhythmias, pulmonary thromboembolism, or severe anemia.  

 
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- 
Have relative contraindications for performing a CPET such as bradyarrhythmias or 
tachyarrhythmias, moderate valvular stenosis, inability to perform physical or mental 
exertion, chronic infectious diseases, musculoskeletal disabilities, ventricular aneurysm, 
second- or third-degree atrioventricular block, or severe arterial hypertension. 
 
3.3 MEASURING INSTRUMENTS 
 INDIRECT RESTING CALORIMETRY: Q-RNG 
Resting calorimetry is an assessment tool that allows us to determine resting energy expenditure 
(REE), RER and some oxidation variables of different energy substrates (57). A study has validated 
this assessment method in adults with different diseases and has proven its effectiveness in 
estimating REE in relation to standard predictive formulas (57). Specifically, it will be performed 
with the Q-RNG (COSMED, Rome, Italy), which has already been validated in various publications 
(61,62,63). 
     BIOCHEMISTRY: AFINION 2 
The Afinion 2 is a multi-assay analyser that provides valuable immediate diagnostic tests (55,56). 
This tool has been validated for measuring glycosylated haemoglobin (HbA1c) and lipid profile 
(TG, total cholesterol, HDL, LDL) in the healthcare systems of Canada and Italy with populations 
with metabolic dysfunction (55). Similarly, a study evaluating the analytical performance of C-
reactive protein (CRP) using the Afinion 2 showed that its results were closer and more similar 
to the central laboratory method, considered the gold standard, than the other evaluation 
methods used in the study (56).  
     INDIRECT CALORIMETRY DURING EXERCISE: GERATHERM ERGOSTIK  
This tool allows the measurement of energy expenditure, CHOox and FATox during the test, among 
other variables, through a gas exchange mask that estimates and predicts the above based on 
oxygen used (VO2) and carbon dioxide expelled (VCO2) (32,33). 
Specifically, the Geratherm Ergostik analyser is based on the breath-by-breath methodology. In 
a study conducted on healthy men, the Ergostik was considered an analyser with good 
reproducibility and reliability when comparing the results obtained on different days (33). 
Another study compared fifteen analysers that allow cardiorespiratory fitness to be assessed, 
including the Geratherm Ergostik. An error of less than 5% was observed between the analysers, 
which were found to be highly accurate (60). 
     CYCLE ERGOMETER: ERGOLINE ERGOSELECT 100 
A cycle ergometer is a lower limb cycling tool that estimates different variables related to power. 
It also allows for the improvement and evaluation of both cardiac and respiratory parameters 
(34).  
Various studies have used a conventional cycle ergometer to evaluate and intervene with 
exercise protocols in populations with different cardiorespiratory and metabolic diseases 
(21,35,36,37).  

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
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     DEXA: HOLOGIC DISCOVERY QDR Wi  
Dual-energy X-ray absorptiometry (DEXA) is an imaging test that mainly allows the evaluation of 
bone mineral density (BMD) for the diagnosis of osteoporosis, and body composition (39).  
Specifically, recent research has demonstrated the accuracy of this DEXA model for measuring 
BMD. However, no significant differences in this variable have been found compared to 
conventional models (39,40).  
     BIOIMPEDANCE: INBODY 770 
Electrical bioimpedance (BIO) is a technique used to estimate body composition through 
different frequencies that travel through the body and indirectly estimate the compartments of 
fat mass, muscle mass and body water, among others (41).  
Various studies have confirmed the reliability of this model for estimating body composition in 
healthy populations to estimate the percentage of fat mass and fat-free mass (41,42). In addition, 
this tool has been used to assess body composition and TMB as well as PhA in a study conducted 
on BC survivors (42) and, in another study, to assess Xc and R in trained athletes (43).  
     NEAR-INFRARED SPECTROSCOPY: NIRS (PortaMon) 
Over the last decade, this tool has been used to determine muscle oxygenation in different 
sporting contexts, thus providing interesting information on the oxidative capacity of the muscle 
(44,45,46,47). Its reliability has been proven in different studies in both trained and untrained 
populations (44,45,46,47,58).  
     LACTATE ANALYZER: LACTATE PLUS 
Blood lactate has been considered an indirect indicator of muscle fibre metabolic function 
(20,21). Thus, some authors have conducted various studies to evaluate blood lactate in 
cardiorespiratory assessment tests in healthy populations (48). This assessment method has 
spread throughout the high-performance sports community, mainly in endurance sports, to 
evaluate certain parameters related to the metabolic function and performance of athletes (49). 
High inter- and intra-device reliability and adequate accuracy have been observed in the Lactate 
Plus device in both humans and murine models (50,59).  
     KFORCE: KINVENT 
Isometric strength will be measured using a pressure dynamometer (KFORCE, KInvent, 
Montpellier, France) that records the kilograms of pressure completed (68,69). This assessment 
tool has been validated in healthy subjects with no history of previous injury to determine the 
isometric strength of the pectoral muscles and the isometric strength of the anterior thigh 
muscles (68,69). 
     HEART RATE MONITOR: POLAR H10  
According to a study conducted with recreational athletes at rest and during an incremental test, 
the H10 heart rate (HR) sensor was shown to be highly reliable and valid for measuring both HR 
and HR variability (HRV) (51).  

 
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     FATIGUE QUESTIONNAIRE: PERFORM 
This questionnaire is the only one validated for determining fatigue in cancer patients (52). Its 
reliability, validity and sensitivity for quantifying improvements or detriments in variables 
associated with fatigue have been proven (52).  
     QUALITY OF LIFE QUESTIONNAIRE: EORTC QLQ-C30 
The European Organisation for Research and Treatment of Cancer (EORT) designed the Quality 
of Life Questionnaire C30 (QLQ-C30), which addresses aspects related to functional health, 
cancer symptoms, and other aspects of quality of life (53). Its validity has been demonstrated in 
patients at all stages of the disease (53).  
     QUALITY OF LIFE QUESTIONNAIRE: EORTC QLQ-BR45 
There is a complementary module to the general questionnaire above depending on the type of 
tumour diagnosed, so patients with BC have an additional section for providing different tumour-
specific responses (54). Some studies have already confirmed its reliability and validity in other 
countries for assessing quality of life in patients with BC (54). 
 
3.4 ANALYSED VARIABLES 
The following variables will be evaluated after diagnosis, at the end of neoadjuvant treatment, 
after recovery from breast surgery, and at the end of the physical exercise programme. 
 Indirect calorimetry at rest: FATox, CHOox, REE, RER.  
 Cycle ergometer CPET: W, CHOox, FATox, VO2máx, RER, ventilatory volume (VE), VO2, VCO2, 
partial pressure of carbon dioxide (PETCO2), ventilatory oxygen equivalent (VE/VO2), 
ventilatory carbon dioxide equivalent (VE/CO2), HR, HRV, watts at maximum fat 
oxidation point (WFATmáx), and lactate in peripheral blood sample.  
 Assessment of muscle oxygenation during cycle ergometer CPET: HbO2, HHb,      %TSI, 
HbO2 resaturation and %TSI resaturation.  
 Body composition: body mass index (BMI), % total fat mass, muscle mass, bone mass, 
BMD, total visceral fat volume (cm3 VAT), TMB, PhA, Xc and R.  
 Biochemical parameters: glucose, lactate, HDL, LDL, TG, total cholesterol, HbA1c, and 
CRP.  
 Metabolic function: takes into account RER, FATox y CHOox, kilocalories (KCAL) from fat 
and KCAL from CHO, and lactate concentration.  
 Isometric strength test of the pectoral muscles and anterior thigh muscles: maximum 
upper limb strength (SmaxUL), maximum upper limb strength time (TSmaxUL), upper 
limb rate of force development (RFD_UL), maximum right lower limb strength (SmaxRL), 
maximum left lower limb strength (SmaxLL), lower limb RFD (RFD_LL), and lower limb 
asymmetry (AsymLL). 
 

 
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3.5 STUDY PROCEDURE 
Prior to the inclusion of patients and their initial assessment, the IC (Annex 1) will be completed 
and formalised in the aforementioned hospitals. Data will be collected from the analyses 
associated with each assessment stage of the descriptive protocol (diagnosis, post-NA, post-
surgery recovery and post-intervention) and from routine analyses containing some of the 
biochemical parameters mentioned above, as requested by the oncology team at their 
respective hospitals. If this is not possible, these parameters will be taken at the European 
University of Madrid (C/ Tajo, s/n, Urbanización El Bosque, Edificio D, 28670 Villaviciosa de Odón, 
Madrid) prior to each assessment.  
Next, DEXA and BIO will be performed to determine the body composition of all patients in the 
study, carried out at the European University of Madrid.  
Similarly, before starting the resting calorimetry and CPET, the EORTC QLQ-C30, EORTC QLQ-BR45 
and FATIGA PERFORM questionnaires related to the quality of life and health of patients with BC 
will be administered at the European University of Madrid.  
Once all of the above has been carried out, resting calorimetry will be performed. This test will 
begin with the patient in a supine position on a examination table and will last approximately 15 
minutes. The chamber surrounding the patient will rest on the mid-abdominal area and the front 
of the shoulders, so that the gases are directed exclusively towards the umbilical region. The 
patient will be instructed to ensure correct gas exchange by not altering their normal breathing, 
not falling asleep and not speaking until the test is complete. Similarly, there will be no external 
disturbances that could interfere with the proper completion of the test. To this end, the room 
will be silent and the ambient temperature will be between 18 and 22 degrees celsius. The test 
will be carried out on the premises of the European University of Madrid by the study 
researchers.  
Once the resting calorimetry has been completed, the isometric strength tests will be carried 
out. Firstly, the pectoral muscle strength test will consist of a 5-second isometric action to 
analyse the neuromuscular capacities of the aforementioned muscles at 90° of shoulder flexion. 
Secondly, the anterior thigh muscle strength test will consist of performing an isometric knee 
extension in a seated position for 5 seconds, starting with a 90° knee flexion. The procedure will 
be repeated 3 times for each test.  
All participating patients will perform an CPET on a cycle ergometer using a incremental stepwise 
protocol to indirectly assess metabolic flexibility through gas exchange. Subsequently, a 
maximum ramp protocol will also be performed on a cycle ergometer to estimate VO2máx. These 
two protocols will be repeated at three key moments in the newly diagnosed BC patients 
selected for the study: before NA treatment; after NA treatment; and after surgery. Patients will 
then enter the physical exercise intervention, either through the CG or the experimental groups 
(E1, E2, or E3), and the final assessment of CPET will be carried out using the aforementioned 
protocols (Figure 1). 
 
 

 
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Figure 1. Longitudinal study procedure.  
 
The CPET will follow a incremental step protocol in which participants will begin with a load of 0 
W for three minutes to stabilise gas exchange data. The load will then be increased based on 
each participant's training level and the previously estimated relative power that takes into 
account the subject's weight (Table 2). To this end, a Pmax value of 175W is estimated for 
untrained subjects, a Pmax value of 245W for physically fit subjects, and a Pmax value of 315W for 
trained subjects (Table 2). At least four three-minute periods representing between 20 and 60% 
of PAM must be completed, where the intensity at which fat oxidation is highest (FATmax) is 
expected to be found. The three-minute per step protocol will be continued until the RER is 
greater than 1. In addition, pedalling must be maintained between 60 and 90 revolutions per 
minute (RPM) during the test. The CPET with the incremental step protocol will have a total 
duration of between 15 and 21 minutes depending on the cardiovascular and metabolic capacity 
of each patient.  
After a 5-minute rest, participants from the stepwise protocol will perform the maximum ramp 
protocol. The test begins with the last load imposed in the step test and will increase by 3 W 
every 12 seconds until the patient is exhausted. It is not possible to know the approximate total 
duration of the CPET with this protocol due to differences in the physical condition of each 
patient. 
 
 
 
 
 
 
 

 
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Table 2. Estimation of MAP based on training level and weight. Example: subject weighing 60 kilograms. 
 
HbA1c, CRP, and lipid profile samples (in cases where hospital test results are not available) will 
be collected once for each complete patient evaluation at the European University of Madrid, 
under resting conditions. Similarly, lactate and glucose samples will be taken at rest before 
starting the CPET; however, this procedure will be repeated at the end of each 3-minute period 
during the stepwise protocol, at the beginning and end of the ramp protocol, and after 3 minutes 
of active recovery without resistance from the ramp protocol (Figure 2). 
 
 
 
 
 
 
 
 
 
MAP estimated (w/kg)
Increase (W/kg/stage)
Subject weight
Pmax
2,92
0,29
60,00
175,00
Stage
Load (W/kg)
Absolut load
%MAP
1,00
0,29
17,50
0.1
2,00
0,58
35,00
0.2
3,00
0,88
52,50
0.3
4,00
1,17
70,00
0.4
5,00
1,46
87,50
0.5
6,00
1,75
105,00
0.6
MAP estimated (w/kg)
Increase (W/kg/stage)
Subject weight
Pmax
4,08
0,41
60,00
245,00
Stage
Load (W/kg)
Absolut load
%MAP
1,00
0,41
24,50
0.1
2,00
0,82
49,00
0.2
3,00
1,23
73,50
0.3
4,00
1,63
98,00
0.4
5,00
2,04
122,50
0.5
6,00
2,45
147,00
0.6
MAP estimated (w/kg)
Increase (W/kg/stage)
Subject weight
Pmax
5,25
0,53
60,00
315,00
Stage
Load (W/kg)
Absolut load
%MAP
1,00
0,53
31,50
0.1
2,00
1,05
63,00
0.2
3,00
1,58
94,50
0.3
4,00
2,10
126,00
0.4
5,00
2,63
157,50
0.5
6,00
3,15
189,00
0.6
Poorly trained
Physically fit
Trained

 
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Figure 2. Measurement of lactate and glucose samples during step and ramp protocols. 
 
 EXERCISE PROGRAM 
With regard to the intervention, there will be three experimental groups (E1, E2, and E3) and a 
CG, resulting from the D1 initial sample. For the assignment of the experimental groups and the 
CG, the D1 group will be randomized. 
On the one hand, the participants in E1 will join a 12-week cardiovascular training program on a 
stationary bike at the W associated with FATmáx, which was determined in the CPET after recovery 
from surgery, two days a week. Thus, there will be two weeks of familiarization in which the 
session will last 60 minutes at the intensity described above. Weeks three and four of the 
intervention will be marked by an increase in volume in minutes while maintaining the intensity, 
with a total session time of 65 minutes. This will be followed by 75 minutes of work at FATmáx W 
over the next eight weeks. To monitor the internal load, we will work at around 3 RPE (Rate of 
Perceived Exertion) throughout the intervention. Finally, the session will end with 10 minutes of 
cool-down with joint mobility exercises and active stretching. Numerous stationary bikes and a 
functional room will be available for the cardiovascular training of group E1 at the European 
University of Madrid. 
On the other hand, participants in E2 will carry out a 12-week cardiovascular training program 
on a stationary bike at MAP, which was determined in the CPET after recovery from surgery, two 
days per week. Thus, during the first two weeks of familiarization, ten 1-minute intervals at MAP 
will be carried out with a 1-minute rest between intervals at 10% of MAP. Subsequently, the 
interval time will be increased in the following two weeks (weeks 3 and 4), with six 2-minute 
intervals at MAP with a 2-minute rest between intervals at 10% of MAP. Finally, four 3-minute 
intervals at MAP will be reached in the remaining eight weeks, with a 3-minute rest between 
intervals at 10% of MAP. To monitor internal load, we will work at around 7-8 RPE throughout 

 
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the intervention. Prior to the main interval training, there will be a 5-minute warm-up at 60% of 
MAP. The session will end with a 10-minute cool-down with joint mobility exercises and active 
stretching. Numerous stationary bikes and a functional room will be available for the 
cardiovascular training of group E2 at the European University of Madrid. 
Both cardiovascular training programs (E1 and E2) will have the same perceived exertion index 
per session (SRPE), considering this to be the RPE at the end of the session, multiplied by the 
total minutes of work. In this way, the total training load per patient will be obtained for each 
session (Table 3).  
Table 3. Total load of the training groups throughout the intervention.  
 
Participants in E3 will follow a 12-week strength training program with progressive loads, twice 
a week. The intervention in this group will be based on the key training principles in the field of 
health: the principle of individualization, the principle of supercompensation, and the principle 
of specificity. 
Each strength training session will begin with joint mobility and muscle activation exercises. The 
latter will consist of core exercises (shoulder girdle, lumbopelvic girdle, and gluteal activation) 
and plyometrics. There will be a two-week familiarization period that will take into account the 
patient's initial level, starting with moderate loads around 6 RPE and a high number of repetitions 
(12) with a high repetitions in reserve (RIR) (4). Between weeks three and four, patients should 
perform 10 repetitions per strength exercise with a moderate RIR (3) and an RPE of 7. The 
following eight weeks will progress to high loads with an RPE between 8-9 and a lower number 
of repetitions (8-6) with a low RIR (2-1), thus attempting to maximize adaptations to training. 
They will start with 3 sets during the first six weeks, reaching 4 sets in the remaining six weeks 
of the intervention (Table 3).  
The exercises to be implemented during the intervention are as follows: two knee-dominant 
lower limb exercises, hex bar squats and front lunges; two hip-dominant lower limb exercises, 
bilateral glute bridge and unilateral hamstring bridge; two upper limb pushing exercises, bench 
press and unilateral landmine in knight position; and two upper limb pulling exercises, neutral 
pull-ups with elastic band and unilateral dumbbell row.  
Table 3. Progression of experimental group 3 (E3) throughout the strength training programme. 
 
Finally, there will be a CG that will not be included in the intervention programs. However, they 
will be informed about general exercise recommendations for cancer patients. Like groups E1, 
E2, and E3, they will be evaluated at the end of the exercise intervention (Figure 1). In addition, 
they will subsequently be offered the program that has obtained the best results in the metabolic 
flexibility parameters and the rest of the variables analyzed.  
 
WORKING TIME
INTERVALS sRPE LOAD
WARP-UP
INTERVAL TIME
NUMBER OF INTERVALS
TIME BETWEEN INTERVALS
NUMBER OF BREAKS sRPE LOAD
1ª/2ª WEEK
60
1
3
180
5
1
10
1
10
7
175
3ª/4ª WEEK
65
1
3
195
5
2
6
2
6
7
203
5ª - 12ª WEEK
75
1
3
225
5
3
4
3
4
8
232
INTERVENTION
E1- CONTINUOUS TRAINING
E2- INTERVAL TRAINING
WEEK 1
WEEK 2
WEEK 3
WEEK 4
WEEK 5
WEEK 6
WEEK 7
WEEK 8
WEEK 9
WEEK 10
WEEK 11
WEEK 12
EXERCISE
SETS
3
3
3
3
3
3
4
4
4
4
4
4
REPETITIONS
12
12
10
10
8
8
8
8
7
7
6
6
RIR
4
4
3
3
2
2
2
2
1
1
1
1
RPE
6
6
7
7
8
8
8
8
9
9
9
9
Hex bar squat, front lunge, bilateral glute bridge, unilateral hamstring bridge, bench press, unilateral landmine, pull-ups and unilateral dumbbell row.

 
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To determine the most effective physical exercise intervention for improving metabolic flexibility 
and metabolic function, the following will be taken into account: the intervention group with the 
highest number of patients who decrease RER and lactate during CPET, defined at the end of 
each step in the stepwise protocol during the evaluation to be carried out after surgery, 
compared to the evaluation to be carried out after the exercise intervention; the intervention 
group with the highest number of patients who increase FATox and CHOox, as well as the 
respective kilocalories (KCAL), at the points defined at the end of each step mentioned, obtained 
from the CPET after surgery, in relation to the results of the CPET performed at the end of the 
exercise intervention; the experimental group with a statistically significant inference in the 
changes produced in the variables mentioned above. 
 ETHICAL AND CONFIDENTIALITY ISSUES 
Informed consent 
All participants will be informed about their inclusion in the study by means of an informed 
consent form, which will explain what their participation in the study entails. 
Voluntary participation 
Participation by patients is, of course, entirely voluntary, and they may decide whether or not to 
participate without this affecting their medical care in any way. 
Data protection 
The Data Controller (the Principal Investigator, Dr Lidia B. Alejo), in compliance with Organic Law 
3/2018 of 5 December on the Protection of Personal Data and the guarantee of digital rights and 
Regulation (EU) 2016/679 of the European Parliament and of the Council of 27 April 2016 on the 
protection of natural persons with regard to the processing of personal data and on the free 
movement of such data, undertakes to accept its confidentiality responsibilities.  
The data will be included in a file owned by UNIVERSIDAD EUROPEA DE MADRID, S.L.U, with 
registered office at C/ Tajo s/n, Villaviciosa de Odón, Madrid, which is the recipient of the 
information provided, for the purpose of being managed by the OTRI – Office for the Transfer of 
Research Results of the European University of Madrid, for the call for external research projects.  
The information obtained will be stored in a computerised database and the data will be 
processed statistically in coded form. Donors will have the right to access, rectify or cancel the 
data stored in the database at any time, provided they expressly request to do so. To do so, they 
must contact the principal investigator. The data will be kept under the responsibility of the 
Principal Investigator of the Study, Dr Lidia B. Alejo. 
Obtaining and using biological samples 
The extraction of glucose, lactate, glycosylated haemoglobin, C-reactive protein, and the lipid 
profile will be carried out by professionals in the field and in an environment that complies with 
the specific regulations for doing so, at the European University of Madrid. The second drop of 
blood will be extracted, discarding the first, for each biochemical parameter by capillary puncture 
of the middle or ring finger of the non-dominant hand, if possible. 

 
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The samples of these biochemical parameters will be used exclusively for the indirect 
determination of metabolic flexibility. The biological samples will be used only for this project 
and will be destroyed at the end of the analysis at the time of collection.  
Risks and discomfort associated with participation in the study 
The discomfort associated with finger pricking refers to mild pain at the time of the prick, redness 
at the puncture site, residual blood droplets after extraction, small bruising, and/or minimal local 
inflammation.  
Strength training may carry a certain risk of joint, tendon, and muscle stress, which increases the 
risk of injury and muscle overload (70,71). Similarly, training with heavy weights may raise blood 
pressure and increase the risk of acute cardiovascular events (70,71).  
Strenuous, intense, prolonged physical exercise or exercise with a high demand for eccentric 
contractions could lead to delayed onset muscle soreness (DOMS) and muscle fatigue, causing a 
temporary loss of maximum voluntary contraction strength (74). 
There may be a high risk of acute cardiovascular events when exercising if the person is sedentary 
and has known or hidden heart disease during high-intensity exercise (72).  
Performing an CPET can cause certain changes such as abnormal blood pressure, fainting, or 
irregular, slowed, or accelerated heart rhythm (73). There is a minimal risk of sudden cardiac 
death, specifically 0 to 5 per 100.000 tests, or 0.005% (73). Similarly, the risk of complications 
requiring hospitalisation (including severe arrhythmias), acute myocardial infarction or sudden 
cardiac death during or immediately after the test is less than 0.2% in the first case and 0.04% 
and 0.01% respectively (73).  
 

 
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and 
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physical 
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 STUDY RESEARCH TEAM 
Table 4. Research team work schedule. 
 
 
 
 
Informed consent
María José 
Echarri, Ana 
Isabel 
Ballesteros, 
Dulce Bañón, 
Berta Obispo 
and nursing 
team
X
X
X
X
X
X
X
X
X
X
X
X
Exercise 
programmes
Adriana Gallo
X
X
X
X
X
X
X
X
X
X
X
Data analysis
Blanca Romero, 
Lidia Brea , Itizar 
Pagola 
Aldazabal, David 
Barranco, 
Adriana Gallo
X
X
X
X
Publication of the 
results
The whole team
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Completion of 
questionnaires, 
resting calorimetry, 
bone densitometry, 
and exercise test
Lidia Brea, Itziar 
Pagola 
Aldazabal, David 
Barranco,  
Adriana Gallo
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Biochemistry
María José 
Echarri, Ana 
Isabel 
Ballesteros, 
Dulce Bañón, 
Berta Obispo 
Andrea Calderón
X
X
X
X
X
X
X
X
X
X
MONTH 23
MONTH 24
Recruitment
 María José 
Echarri, Ana 
Isabel 
Ballesteros, 
Dulce Bañón, 
Berta Obispo 
and nursing 
team
X
X
X
X
X
X
MONTH 17
MONTH 18
MONTH 19
MONTH 20
MONTH 21
MONTH 22
MONTH 11
MONTH 12
MONTH 13
MONTH 14
MONTH 15
MONTH 16
MONTH 5
MONTH 6
MONTH 7
MONTH 8
MONTH 9
MONTH 10
Task
Involved and 
responsible 
members
MONTH 1
MONTH 2
MONTH 3
MONTH 4

 
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 PATIENT TIMELINE 
Table 5. Example of a schedule for a patient recruited in the study.   
 
DEXA: bone densitometry; E1: cardiovascular training group around FATmáx; E2: cardiovascular training group around MAP; E3: strength training group; CG: control group.
W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4
E1, E2, E3, CG
RECRUITMENT AND INFORMED CONSENT
BIOCHEMISTRY RECORD
IMPLEMENTATION OF QUESTIONNAIRES, RESTING CALORIMETRY, DEXA, EXERCISE TEST
NEOADYUVANT TREATMENT
SURGERY
CARDIOVASCULAR INTERVENTION GROUP (E1)
CARDIOVASCULAR INTERVENTION GROUP (E2)
STRENGHT INTERVENTION GROUP (E3)
MONTH 7
MONTH 8
MONTH 9
MONTH 10
MONTH 11
MONTH 12
MONTH 1
MONTH 2
MONTH 3
MONTH 4
MONTH 5
MONTH 6

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
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 STUDY BUDGET 
It is estimated that between 9 and 10 lactate strips will be used per patient per assessment, 
depending on the number of steps required to complete the stepwise protocol. Therefore, 
considering that four assessments with lactate collection will be carried out throughout the 
study, 36 lactate strips will be used per patient. The initial sample will be 40 patients, therefore 
the total number of lactate strips is 1,440, which multiplied by the individual cost of €3, gives a 
total value of €4,320.  
It is estimated that between 9 and 10 glucose strips will be used per patient per assessment, 
depending on the number of steps required to complete the stepwise protocol. Therefore, 
considering that four assessments with glucose collection will be carried out throughout the 
study, 36 lactate strips will be used per patient. The initial sample will be 40 patients, so the total 
number of glucose strips is 1,440, which multiplied by the individual cost of €0.28, gives an 
estimated total value of €403.20.  
One lipid profile per patient per assessment will be used, and considering that four assessments 
will be carried out throughout the study, four lipid profiles per patient will be used. This 
multiplied by the 40 patients in the sample equals a total of 160 lipid profiles. Thus, 160 
multiplied by the cost of each lipid profile of €9 gives a total value of €1,440. 
One HbA1c test per patient per assessment will be used, and, considering that they will undergo 
four assessments throughout the study, four HbA1c tests per patient will be used. Multiplying 
this by the 40 patients in the sample gives a total of 160 HbA1c tests. Thus, 160 multiplied by the 
cost of each HbA1c analysis of €6 gives a total value of €960.  
One CRP analysis per patient per assessment will be used, and considering that they will undergo 
four assessments throughout the study, four CRP analyses per patient will be used. This 
multiplied by 40 patients in the sample gives a total of 160 CRP analyses. Thus, 160 multiplied 
by the cost of each CRP analysis of €6 gives a total value of €960.  
One resting calorimetry test will be performed per patient per assessment, with four 
assessments throughout the study. Therefore, 4 resting calorimetries will be performed per 
patient during the research. Considering 40 patients as the initial sample, 160 resting 
calorimetries will be performed, which multiplied by the cost of €80, gives a total value of 
€12,800. 
Two CPET will be performed per patient for each evaluation, with four evaluations throughout 
the study. Thus, eight CPET will be performed per patient during the investigation. Considering 
40 patients as the initial sample, 320 CPET will be performed, which multiplied by the cost of the 
test of €145, reports a total value of €46,400.  
Finally, one DEXA scan will be performed per patient for each assessment, with four 
measurements throughout the study. Thus, four DEXA scans will be performed per patient during 
the research. Considering 40 patients as the initial sample, 160 DEXA scans will be performed, 
which multiplied by the cost of the test of €120, reports a total value of €19,200.  
Accident insurance for participants: 

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
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The cost of accident insurance per patient is €12. Taking into account the initial sample of 40 
participants, €480 would be required.  
Civil liability insurance: 
The cost of civil liability insurance is €2,558.57.  
Travel and subsistence expenses: 
Travel between the different hospitals and the University will be required, for which the 
estimated cost is €800. 
Publication and dissemination of results: 
The aim is to publish at least two articles in Q2-Q1 journals, based on the team's previous 
experience in similar research. One of these articles is intended to be published in an Open 
Access (Q1) journal in the field of Sport Science, Physiology or Oncology, for which an associated 
budget is required. The estimated cost for this item is €2,100 (Table 6). 
Table 6. Study Budget.  
 
 
3.6 STATISTICAL ANALYSIS 
The data will be processed and analysed using IMB SPSS Statistics version 29.0 (International 
Business Machines Corporation, Armonk, USA). Descriptive statistics will be expressed as 
percentages, means and standard deviations (SD) for all variables to be analysed. To analyse the 
distribution of all variables, the Kolmorgorov-Smirnov normality test will be performed before 
conducting inferential statistics. Thus, if the variable is parametric and follows a normal 
distribution, Student's t-tests or ANOVA will be used, and Pearson's correlation will be used to 
determine the relationships between two variables. On the other hand, if the variable is non-
parametric and does not follow a normal distribution, Wilcoxon's or Mann-Whitney's U tests will 
be used, and Spearman's correlation will be used to determine the relationships between two 
variables.  
Test
Quantity
€/ unit
€Total 
Lactate strips
1440
3
4320
Glucose strips
1440
0,28
403,2
Lipid profile
160
9
1440
HbA1c
160
6
960
CRP
160
6
960
Resting calorimetry
160
80
12800
Cardiopulmonary exercise test
320
145
46400
DEXA
160
120
19200
Accident insurance 
40
12
480
Civil liability insurance
1
2558,57
2558,57
Travel and subsistence expenses
1
800
800
Publication of results
1
2100
2100
TOTAL
92421,77

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
Version 2. 17-11-2025.                                                                                                                                                                     PI: Lidia B. Alejo 
 
During the statistical analysis, an effect size (ES) according to Cohen's d will be taken into account. 
To this end, a weak ES will be established with values between 0.2 and 0.5, a medium ES with 
values between 0.5 and 0.8, and a strong ES with values above 0.8. For all statistical analysis, the 
significance level will be p ≤ 0.05.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
Version 2. 17-11-2025.                                                                                                                                                                     PI: Lidia B. Alejo 
 
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740. Disponible en: http://dx.doi.org/10.1161/hc3901.095960 
77. Paulo AC, Forjaz CLM, Mion D Jr, Silva GV, Barros S, Tricoli V. Blood pressure increase in hypertensive 
individuals during resistance training protocols with equated work to rest ratio. Front Physiol [Internet]. 
2020;11:481. Disponible en: http://dx.doi.org/10.3389/fphys.2020.00481 
78. Thompson PD, Arena R, Riebe D, Pescatello LS, American College of Sports Medicine. ACSM’s new 
preparticipation health screening recommendations from ACSM’s guidelines for exercise testing and 
prescription, ninth edition. Curr Sports Med Rep [Internet]. 2013;12(4):215–7. Disponible en: 
http://dx.doi.org/10.1249/JSR.0b013e31829a68cf 
79. Williams MA, Haskell WL, Ades PA, Amsterdam EA, Bittner V, Franklin BA, et al. Resistance exercise in 
individuals with and without cardiovascular disease: 2007 update: a scientific statement from the American 
Heart Association Council on Clinical Cardiology and Council on Nutrition, Physical Activity, and Metabolism: 
A scientific statement from the American Heart Association council on clinical cardiology and council on 
nutrition, physical activity, and metabolism. Circulation [Internet]. 2007;116(5):572–84. Disponible en: 
http://dx.doi.org/10.1161/CIRCULATIONAHA.107.185214 
 
 
 
 
 

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
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APPENDICES 
APPENDIX 1 
INFORMATION SHEET 
Study entitled: “Metabolic flexibility in patients with early triple-negative breast cancer and the 
possible effect of a physical exercise intervention” 
You are being asked to participate in a study entitled: “Metabolic flexibility in patients with early 
triple-negative breast cancer and the possible effect of a physical exercise intervention”. This 
research has been approved by the Research Ethics Committee of Getafe University Hospital. 
Voluntary participation 
The healthcare personnel treating you have proposed that you participate in a study to 
determine the metabolic flexibility of muscle fibres, i.e., to verify the substrate with the highest 
preference in muscle fibre and the existence or absence of mitochondrial dysfunction due to 
cancer treatments and the pathophysiology of the disease. In addition, the aim is to find out 
whether the proposed physical exercise programmes can modify the aforementioned metabolic 
flexibility. Your participation is, of course, entirely voluntary and you can decide whether or not 
to participate without this affecting your medical care in any way.  
General information 
Some authors suggest that physical exercise has a significant influence on glucose levels, insulin 
resistance, growth factors, fat oxidation ratio, and lactate, among other factors. Alteration of any 
of these markers could activate tumour signalling pathways, thus representing a risk factor for 
tumour proliferation. Therefore, it could be concluded that the modulation of metabolism 
through physical exercise plays a fundamental role in the development of cancer.  
Physical exercise is considered a key tool for improving metabolic flexibility in muscle fibre. In 
response to increased AMPK and PGC-1α activity with training, increases in mitochondrial 
content and function have been observed in muscle fibre. This has a positive effect on the ability 
to oxidise fatty acids in the mitochondria, improving insulin sensitivity. The above would improve 
the ability of muscle fibre, now with a greater number of mitochondria, to oxidise different 
substrates, thus improving metabolic flexibility and adapting to different energy requirements 
with activity.  
Purpose of this study 
This study has two main objectives: 
a) To describe the metabolic function of women diagnosed with triple-negative breast 
cancer at different stages of early disease.  
b) To verify the effects of different training interventions on the metabolic function of 
women diagnosed with triple-negative breast cancer.  
Description of the tests 

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
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Your participation in the study will consist of undergoing the following assessment tests, which 
will be repeated four times throughout the study (after diagnosis, at the end of neoadjuvant 
cancer treatment, after surgery, and at the end of the physical exercise intervention): a 
biochemical test if you have not had one done at the hospital; a DEXA scan; an electrical 
bioimpedance test; three questionnaires, EORTC QLQ-C30, EORTC QLQ-BR45 and FATIGA 
PERFORM, related to quality of life and fatigue; a resting calorimetry test; two isometric strength 
tests of the pectoral muscles and the anterior thigh muscles; a cycle ergometer CPET using an 
incremental methodology; and a CPET using a ramp protocol. The complete assessment includes 
all of the above tests and will be carried out on the same day over approximately two hours for 
each patient at the European University of Madrid. 
Following the patient's surgery, there will be three experimental groups (E1, E2, E3) and a control 
group (CG). If randomly selected for the experimental group, they will participate in moderate 
cardiovascular training two days per week, interval cardiovascular training two days per week, 
or strength training two days per week at the European University of Madrid. 
On the one hand, participants in E1 will join a 12-week cardiovascular training programme on a 
stationary bike at the W associated with FATmáx, which was determined in the incremental 
exercise test after recovery from surgery, two days a week. There will be two weeks of 
familiarisation, during which the session will last 60 minutes at the intensity described above. 
Weeks three and four of the intervention will be marked by an increase in volume in minutes 
while maintaining the intensity, with a total session time of 65 minutes. This will be followed by 
75 minutes of work at the W associated with FATmáx over the next eight weeks. Finally, the session 
will end with 10 minutes of cool-down with joint mobility exercises and active stretching. There 
will be numerous exercise bikes and a functional room for cardiovascular training for group E1 at 
the European University of Madrid. 
On the other hand, participants in E2 will carry out a 12-week cardiovascular training programme 
on a stationary bicycle at MAP, which was determined in the ramp exercise test after recovery 
from surgery, two days a week. Thus, during the first two weeks of familiarisation, ten 1-minute 
intervals at MAP will be carried out with a 1-minute rest between intervals at 10% of MAP. 
Subsequently, the interval time will be increased in the following two weeks (weeks 3 and 4), 
with six 2-minute intervals at MAP with a 2-minute rest between intervals at 10% of MAP. Finally, 
four 3-minute intervals at MAP will be reached in the remaining eight weeks, with a 3-minute 
rest between intervals at 10% of MAP. The session will end with a 10-minute cool-down with 
joint mobility exercises and active stretching. There will be numerous exercise bikes and a 
functional room for the cardiovascular training of group E2 at the European University of Madrid. 
Participants in E3 will follow a 12-week strength programme with progressive loads, training 
twice a week. Each strength session will begin with joint mobility and muscle activation exercises. 
This activation exercises will be consisting of core exercises (shoulder girdle, lumbopelvic girdle, 
and gluteal activation) and plyometrics. There will be a two-week familiarisation period in which 
the patient's initial level will be taken into account, starting with moderate loads around 6 on 
the RPE and a high number of repetitions (12) with a high repetitions in reserve (RIR) (4). 
Between weeks three and four, patients should perform 10 repetitions per strength exercise with 
a moderate RIR (3) and an RPE of 7. The following eight weeks will progress to high loads with 
an RPE between 8-9 and a lower number of repetitions (8-6) with a low RIR (2-1), thus attempting 

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
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to maximise adaptations to training. They will start with 3 sets during the first six weeks, reaching 
4 sets in the remaining six weeks of the intervention.  
The exercises to be implemented during the strength intervention are as follows: two knee-
dominant lower limb exercises, hex bar squats and front lunges; two hip-dominant lower limb 
exercises, bilateral glute bridge and unilateral hamstring bridge; two upper limb pushing 
exercises, bench press and unilateral landmine in knight position; and two upper limb pulling 
exercises, neutral pull-ups with elastic band and unilateral dumbbell row.  
Finally, there will be a CG that will not be included in the intervention programmes. However, 
they will be informed about the general exercise recommendations for cancer patients. Like 
groups E1, E2 and E3, they will be evaluated at the end of the exercise intervention. In addition, 
they will subsequently be offered the programme that has obtained the best results in the 
metabolic flexibility parameters and the rest of the variables analysed.  
 
The incremental exercise test will be carried out by exercise professionals (physical education 
and sports instructors) and in the presence of a doctor whenever there is a medium or high risk 
of cardiovascular events, although it is unusual for any type of setback to occur during these 
tests. 
The exercise sessions will be supervised and designed by physical education instructors, meaning 
registered professionals who have completed a degree in Physical Activity and Sports Sciences. 
Collection and use of biological samples 
Glucose, lactate, glycosylated haemoglobin, C-reactive protein and lipid profile tests will be 
carried out by professionals in the field and in an environment that complies with the specific 
regulations for their performance, at the European University of Madrid. The second drop of 
blood will be extracted, discarding the first, for each biochemical parameter by capillary puncture 
of the middle or ring finger of the non-dominant hand, if possible. 
The samples of these biochemical parameters will be used exclusively for the indirect 
determination of metabolic flexibility. The biological samples will be used solely for this project 
and will be destroyed at the end of the analysis at the time of collection.  
Anticipated risks 
The discomfort associated with capillary puncture refers to mild pain at the time of puncture, 
redness at the puncture site, residual blood drop after extraction, small bruising, and/or minimal 
local inflammation.  
Strength training may pose a certain risk of joint, tendon and muscle stress, which increases the 
risk of injury and overload (Chen et al., 2021; Kambič et al., 2024). Similarly, training with heavy 
weights could raise blood pressure and increase the risk of acute cardiovascular events (Chen et 
al., 2021; Kambič et al., 2024).  
There may be a high risk of acute cardiovascular events when exercising if the person is sedentary 
and has known or hidden heart disease during high-intensity exercise (Riebe et al., 2015).  

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
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Strenuous, intense, prolonged physical exercise or exercise with a high demand for eccentric 
contractions could lead to delayed onset muscle soreness (DOMS) and muscle fatigue, causing a 
temporary loss in maximum voluntary contraction strength (Dupuy et al., 2018). 
Under conditions of prolonged fasting or a high contribution of glucose to energy generation 
during exercise, there is a certain risk of hypoglycaemia (Davis et al., 2014). However, this risk 
occurs mainly in diabetic patients (Davis et al., 2014). 
Performing a CPET can cause certain changes such as abnormal blood pressure, fainting, or 
irregular, slowed, or accelerated heart rate (Thompson et al., 2013). There is a minimal risk of 
sudden cardiac death, specifically 0 to 5 per 100,000 tests, or 0.005% (Thompson et al., 2013; 
Myers et al., 2009). Similarly, the risk of complications requiring hospitalisation (including severe 
arrhythmias), acute myocardial infarction, or sudden cardiac death during or immediately after 
the test is less than 0.2% in the first case and 0.04% and 0.01%, respectively (Myers et al., 2009).  
Patients with absolute contraindications for CPET, such as heart failure, myocarditis, acute 
pericarditis, severe aortic stenosis, aortic dissection, vascular toxicity, severe uncontrolled 
hypertension, severe uncontrolled cardiac arrhythmias, pulmonary thromboembolism, or severe 
anaemia, may not be included in the study (Fletcher et al., 2001).  
Similarly, patients with relative contraindications for CPET, such as bradyarrhythmias or 
tachyarrhythmias, moderate valvular stenosis, inability to perform physical or mental exertion, 
chronic infectious diseases, musculoskeletal disabilities , ventricular aneurysm, second- or third-
degree atrioventricular block, or severe hypertension (Fletcher et al., 2001). 
Finally, there are some absolute contraindications for strength training. These include 
uncontrolled hypertension, unstable cardiovascular disease, and acute musculoskeletal injuries. 
Relative contraindications for this type of training include chronic kidney disease, moderate 
hypertension, diabetes mellitus, and proliferative diabetic retinopathy (Thompson et al., 2013; 
Williams et al., 2007; Paulo et al., 2020).  
Duration of the study  
The duration of the longitudinal study will be a maximum of 2 years, including the descriptive 
phase and the physical exercise intervention phase, depending on the protocol followed by each 
patient in their respective hospital in relation to the cancer treatment plan, planned surgery, etc.  
The cardiovascular exercise interventions and strength training intervention will have a total 
duration of 12 weeks, with a training frequency of 2 days per week and a recovery period of at 
least 48 hours between sessions.  
Confidentiality and Data Protection 
The Data Controller (Principal Investigator, Dr Lidia B. Alejo), in compliance with Organic Law 
3/2018 of 5 December on the Protection of Personal Data and the guarantee of digital rights and 
Regulation (EU) 2016/679 of the European Parliament and of the Council of 27 April 2016 on the 
protection of natural persons with regard to the processing of personal data and on the free 
movement of such data, undertakes to accept its confidentiality responsibilities.  

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
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The data will be included in a file owned by UNIVERSIDAD EUROPEA DE MADRID, S.L.U, with 
registered office at C/ Tajo s/n, Villaviciosa de Odón, Madrid, which is the recipient of the 
information provided, for the purpose of being managed by the OTRI – Office for the Transfer of 
Research Results of the European University of Madrid, for the call for external research projects.  
The information obtained will be stored in a database, in computer format, and the data will be 
processed statistically in a coded form. At any time, the donor shall have the right to access, 
rectify or cancel the data stored in the database, provided that they expressly request it. To do 
so, they must contact the principal investigator. The data will be kept under the responsibility of 
the Principal Investigator of the Study, Dr Lidia B. Alejo. 
Insurance 
In accordance with current legislation, you will be insured under a policy taken out by the study 
sponsor to cover any potential risks that may arise from your participation in the study. 
At the start of the study, accident insurance will be taken out for the participants. In addition, all 
members of the research team are registered with their respective professional associations, 
which means that they all have civil liability insurance covering their professional activities. This 
insurance is associated with their professional work and covers the institution responsible for 
the project.  
On the one hand, in the event of an accident, immediate attention must be given, the accident 
must be recorded and reported in the policy's group register, Mapfre must be notified of the 
accident as soon as possible, and the patient's details and documentation relating to the accident 
must be provided. This policy covers all types of accidents that occur during the insured person's 
professional activity within the insured group. In turn, accident insurance covers accidental 
death (€12,000) and permanent disability according to a scale of up to €12,000. The maximum 
limit per claim for all insured persons is €1,500,000. 
On the other hand, civil liability insurance covers everything that may be claimed from the 
insured as a result of damages caused unintentionally to clients and third parties due to events 
arising from their activity, such as: Metabolic flexibility in patients with early triple-negative 
breast cancer and the possible effect of a physical exercise intervention.  
Costs 
Participating in this project will not incur any costs. 
You will be provided with a copy of this information sheet and your informed consent form to 
keep. 
 
The researcher providing this information is: 
 
 
RESEARCHER                                           TELEPHONE NUMBER 

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
Version 2. 17-11-2025.                                                                                                                                                                     PI: Lidia B. Alejo 
 
INFORMED CONSENT 
I, Mr/Mrs/Ms.………………………………………………………………………………………… 
ID/Passport No.……………………………Date of birth……………………………….. 
Address (Street/Road)………………………………………………………………….. 
Town/City……………………………..Postcode……………………………... 
Country…………………………………….Email address…………………………………… 
 
I declare that: 
1. I have understood the information contained in the information sheet for the research 
project “Metabolic flexibility in patients with early triple-negative breast cancer and the 
possible effect of a physical exercise intervention” that has been given to me, and I have 
had the opportunity to resolve any questions I may have had about participating in this 
study. 
2. The personal data collected in the study will be used solely for the purposes of the 
aforementioned project. 
3. I may withdraw from the study at any time: 
● Whenever I wish. 
● Without having to provide any explanation. 
● Without this having any negative repercussions. 
 
We inform you that your personal data will be incorporated into a file for which Dr Lidia Brea 
Alejo is responsible, in order to provide you with the service that is the subject of this 
communication. If you wish to exercise your rights of access, rectification, cancellation or 
opposition, you may contact her by email at lidia.brea@universidadeuropea.es 
We inform you that you may access, rectify, oppose and/or cancel the personal data in our 
databases at any time. To do so, you must contact the person responsible for the file and send 
us a signed request to the address given in the previous paragraph. 
We also inform you that recordings or photographs are occasionally taken and used as evidence 
to justify the project, in which you may appear as a participant. By signing this document, you 
give your express consent to this. 
Data protection: 
☐ I consent to the processing of my personal data in accordance with the information provided.  
Image rights authorisation: 
☐ I consent to the taking, collection and processing of images and/or recordings in accordance 
with the information provided. 
☐ I do not consent to the taking, collection and processing of images and/or recordings in 
accordance with the information provided. 

 
Metabolic flexibility in patients with early triple-negative breast cancer and the possible effect of a physical exercise intervention. 
Version 2. 17-11-2025.                                                                                                                                                                     PI: Lidia B. Alejo 
 
 
I wish to give my consent to participate in the study entitled “Metabolic flexibility in patients 
with early triple-negative breast cancer and the possible effect of a physical exercise 
intervention” and, to this end, I sign this informed consent document in duplicate.  
 
  
 
     
    SIGNATURE OF THE PARTICIPANT/LEGAL REPRESENTATIVE 
 
    At ……..on the……day of……...in 202… 
 
 
 
     
 
   Signature: …………………………. 
 
I have discussed this project with the patient in understandable language. I believe that I have 
informed the patient of the nature of the study and that the patient has understood this 
explanation. I have provided a copy of the information sheet. 
 
 
   SIGNATURE OF THE PERSON RESPONSIBLE FOR THE STUDY 
 
    At ……..on the……day of……...in 202… 
 
 
 
 
 
    Signature: ………………………….