Impact of a Mediterranean Diet (gReenheArTediEt - RATE) on Cardiovascular Disease Risk Factors: a Randomized Clinical Trial.
Phase: NA
Conditions: Cardiovascular Disease Risk
Sponsor: Universidade Católica Portuguesa

Title: Impact of a Mediterranean-type diet (gReenheArTediEt - RATE) on cardiovascular disease
risk factors: a randomized clinical trial.
Project nº: 22/2023/CEFCM
Porto, September 2023

PROTOCOL
Title: Impact of a Mediterranean-type diet (gReenheArTediEt - RATE) on cardiovascular disease risk
factors: a randomized clinical trial.
Introduction
Cardiovascular disease (CVD) encompasses various disorders of the heart and blood vessels1,
with a underlying atherosclerotic process (coronary artery disease, cerebrovascular disease, aortic and
arterial diseases including hypertension and peripheral vascular disease), or in its absence, congenital
heart disease, rheumatic heart disease, cardiomyopathy, and cardiac arrhythmias would be highlighted2.
CVD continues to be identified as the leading cause of death worldwide. It is estimated that in the year
2019, approximately 17.9 million individuals died from this non-communicable disease, accounting for
32% of all global deaths1. In that same year, CVD mortality reached 4.1 million individuals in Europe
(1.9 million deaths in males and 2.2 million deaths in females), representing an increase of two hundred
thousand deaths annually compared to 20173,4. Among CVDs, coronary artery disease and stroke are
listed as the first and second leading causes of death in Europe3. In Portugal, a similar trend is observed
regarding the high incidence of CVD mortality (29.9%); however, unlike other European countries where
the leading cause of death is coronary artery disease, stroke is indicated as the incident with the highest
prevalence at the national level5.
It is considered that CVD is the result of the interaction between genetic predisposition (non-
modifiable risk factors) and environmental influences (modifiable risk factors). Environmental factors
predominate over others and often lead to negative outcomes through the adoption of poor lifestyles, such
as unbalanced diet, physical inactivity, smoking, and excessive alcohol consumption. These are
considered risk factors with direct effects on CVD pathogenesis and indirectly contribute to an increased
risk for chronic diseases, such as hypertension, dyslipidemia, overweight, obesity, and diabetes mellitus2,6.
In Portugal, overweight and obesity are the risk factors with the highest prevalence in the population
(62.1%). Regarding metabolic risk factors, hypertension and high low-density lipoprotein cholesterol
(LDL-c) have a prevalence of 43.1% and 31.5%, respectively7.
In 2004, Yusuf et al. demonstrated, in a randomized trial, the effects of environmental risk factors
(smoking, diet, and physical exercise) and metabolic risk factors (diabetes mellitus, obesity, and
hypertension) on the contribution to myocardial infarction (MI), one of the outcomes of coronary artery
disease. Participants who had a high daily intake of fruits and vegetables and regularly engaged in
physical exercise had a significantly lower risk of MI8. According to the Global Burden of Disease study
in 2017, the dietary habits of the Portuguese population were the third leading risk factor contributing to
the loss of healthy years of life, particularly due to metabolic diseases, circulatory system disorders, and
neoplasms. Other dietary risk factors, such as alcohol consumption, high plasma glucose, high body mass
index (BMI), hypertension, and high LDL-c also contribute significantly to the loss of healthy years of
life. Within dietary habits, low consumption of whole grains, low consumption of fruits, and low
consumption of nuts and seeds were highlighted as the three main risk factors for this loss, suggesting the
importance of modifying comprehensive lifestyle risk factors to prevent CVD9.

Currently, the scientific literature has demonstrated the importance of diet in preventing CVD
and how it can minimize its risk factors. Dietary Approaches to Stop Hypertension (DASH), the
Mediterranean dietary pattern, vegetarian diet, EAT-Lancet diet, among other dietary strategies, have been
published in various scientific journals10-16. The Seven Countries Study, conducted in the 1960s, stands
out, as the revisitation of the Mediterranean dietary pattern identified important beneficial effects in
preventing various non-communicable chronic diseases17-20. This dietary pattern is characterized by a high
intake of fruits, vegetables, legumes, whole grains, nuts, and olive oil as the main source of fat. In
moderation, there is consumption of dairy products, white meats, eggs, and fish, and finally, low
consumption of red meats, processed meats, sugars, and sugary products21. A recent systematic review
and meta-analysis of observational studies found that individuals diagnosed with metabolic syndrome
who had a higher adherence to the Mediterranean diet (MD) exhibited lower triglyceride plasma
concentration and smaller waist circumference, as well as higher levels of high-density lipoprotein (HDL)
cholesterol22. In addition to the significant reduction in triglyceride levels, weight loss and decreased
insulin resistance are associated with high adherence patterns23. Other studies and systematic reviews also
corroborate the relevance of MD for cardiovascular health14,24,25.
Other dietary patterns that have garnered interest within the scientific community due to the accumulation
of scientific evidence on their environmental benefits and potential in preventing non-communicable
chronic diseases are plant-based diets, primarily due to: a) early satiety resulting from increased dietary
fiber intake, b) improved insulin sensitivity due to weight loss, c) low intake of cholesterol and saturated
fatty acids, among others13. However, the methodology for defining "plant-based diet" or "vegetarian
diet" is not consistent, which hinders the comparison of studies and, consequently, the corroboration of
results. Regarding these dietary patterns, favorable risk factors for CVD have been associated, such as
lower blood pressure levels, lower total cholesterol (TC) and LDL cholesterol levels, decreased BMI and
body fat, as well as a decreased risk of diabetes mellitus26-31. On the other hand, some scientific studies
present contradictory results, emphasizing the importance of conducting more studies for robust
conclusions32.
A randomized clinical trial evaluated the improvement of cardiovascular risk profile through the
implementation of an ovo-lacto-vegetarian diet and the Mediterranean diet (MD). Both diets were
effective in weight loss and reduction of body fat, with no significant differences observed. However, the
plant-based diet showed better results in decreasing LDL cholesterol, while the MD led to lower
triglyceride levels33. Another study with a similar purpose found that a low-fat vegan diet improved body
weight, lipid concentrations, and insulin sensitivity compared to the MD. In this study, blood pressure
decreased in both diets, but to a greater extent in the MD34.
Currently, alongside health concerns, another topic that has received significant attention from the
cientific community is the environmental degradation derived from current food production. With the
Projected global population growth reaching 10 billion by 2050, the effects of food production on
greenhouse gas emissions, nitrogen and phosphorus pollution, biodiversity loss, and water and land use
are expected to reach such levels that will destabilize the Earth's system.
Considering that diet has both environmental value and the potential to promote cardiovascular health, the
RATE aims to explore more sustainable dietary options that promote both health and environmental

sustainability. The intervention will be based on the principles of the Mediterranean diet, with a higher
consumption of plant-based sources compared to other mentioned dietary patterns. Additionally, it will
incorporate the benefits of chronobiology, which are important for weight and adiposity control.
Objectives
The aim is to compare the intervention and control groups, after the nutritional and physical exercise
intervention, in the following aspects:
- Quantify the effect of nutritional and physical activity intervention on improving important
cardiovascular disease risk factors: body weight, body composition (% body fat), and waist circumference.
As a secondary objective, we aim to compare the intervention and control groups regarding the following
variables:
- Characterize the lipid profile, inflammatory markers, and glycated hemoglobin (HbA1c);
- Evaluate cardiovascular risk and health;
- Assess adherence to the Mediterranean-type diet;
- Evaluate body composition;
- Evaluate quantitative and qualitative muscle component and muscle performance (frailty, hand grip
strength, and sarcopenia).
To achieve this objective, a randomized clinical study will be conducted with the following tasks:
Task 1 - Recruitment of Participants
Task 2 – Intervention
Task 3 - Evaluation of the impact of intervention
Task 3.1 - Anthropometric Measurements
Task 3.2 - Analysis of Blood Biomarkers
Task 3.3 - Quantitative muscle component and the qualitative and muscular performance
Task 3.4 - Adherence to the dietary regimen
Task 3.5 - Evaluation of quality of life
Task 3.6 - Evaluation of hand grip strength
Task 3.7 - Evaluation of cardiovascular risk level
Methodology
This project will be carried out at the Algarve Biomedical Center – Research Institute (ABC-RI)
and at the University of Algarve, Gambelas campus.
Task 1 - Recruitment of Participants
This project consists of a randomized clinical trial, whose target population is adult volunteers
at risk of cardiovascular disease. Participants will be recruited among men and women aged 50
years or older, who are autonomous in daily life activities, with a cardiovascular disease risk
classification according to SCORE 2 and SCORE2 O.P, and who do not meet any of the
exclusion criteria (Acute Myocardial Infarction occurrence, being under nutritional counseling

by a nutritionist; adopting a vegetarian or strict vegetarian dietary pattern and/or undergoing
nutritional supplementation).
Individuals will be invited to participate voluntarily in this study, receiving detailed information
about the research to allow for an informed and free decision. Individuals who agree to
participate will sign an informed consent.
Task 2 – Intervention
The nutritional intervention under study was designed based on the principles of the
Mediterranean dietary pattern. However, the gReenhAerTediEt - RATE was developed with the
aim of optimizing health, specifically cardiovascular health parameters, while also considering
food sustainability (principles of the Mediterranean pattern and the Eat-Lancet Diet).
The intervention group will be asked to have three (breakfast, lunch, and dinner) to four
(breakfast, lunch, afternoon snack, and dinner) meals per day, adjusting the number of meals
according to their daily routines. Participants will be encouraged not to count calories,
promoting an emphasis on moderation. Regarding the main meals, legumes will be encouraged
as the protein source for dinner. For lunch, the protein component may include white meats, fish,
or eggs. Red and/or processed meats will be excluded in this intervention. Additionally, a
minimum of 12 hours of overnight fasting will be encouraged for all participants in this group.
In the initial phase, a two-hour group session titled "Discovering New Flavors" will be
conducted. The aim of this session is to promote the consumption of healthy foods that should
be included in participants' daily diet. Oat flakes, high-fiber bread varieties, whole-grain
crackers, corn tortillas, hummus, and guacamole will be presented as suggestions for spreads or
to be consumed with vegetable sticks. In order to create a different dynamic and increase
interaction, some recipe suggestions will be prepared together as a group (such as lentil curry,
beetroot hummus, and guacamole). The intention of this activity is also to simplify and
demonstrate how easy it is to prepare these types of recipes.
Subsequently, the participants will start individual nutritional counseling. For the first
consultation, participants will be asked to keep a three-day non-consecutive food diary (i.e., two
weekdays and one weekend day) on paper in advance. Sociodemographic data, biochemical
data, personal and family history, pharmacotherapy, and physical activity practices will be
provided by the A3-COR project, in which the present study is included. In this first
consultation, dietary intake will be assessed through the analysis of the food diary, while
subsequent consultations will involve a 24-hour dietary recall. Anthropometric evaluation will
include parameters such as triceps and biceps skinfold thickness, arm circumference, and arm

muscle area. Other anthropometric measurements will also be provided by the A3-COR project.
After assessing the nutritional status, nutritional counseling will follow the aforementioned
guidelines. Objectives to be achieved by the participants are defined for each phase of the
intervention (Table 1). Lastly, in the first consultation, a recipe book, a leaflet with the discussed
dietary recommendations, and other guidance documents on food sustainability will be provided.
Task 3 - Evaluation of the impact of intervention
To investigate the impact of the nutritional intervention, participants will be evaluated at two
established time points: at the beginning (day 1) and at the end of the intervention period (day
90). This evaluation will be conducted by healthcare professionals and will consist of collecting
a 3-day non-consecutive food diary, administering two validated questionnaires to assess
sarcopenia and frailty, and finally, evaluating the biochemical profile. At an intermediate point
(between days 40 and 50), a dietary adherence questionnaire (MEDAS) will be administered via
telephone.
Task 3.1 - Anthropometric Measurements
Anthropometric measurements, obtained according to standardized protocols, include the
measurement of height, body weight, waist circumference (WC), bicipital and tricipital
skinfolds, and collection of body composition data. Height and weight will be measured with a
stadiometer and a scale, respectively, with participants barefoot. Waist circumference will be
measured at the midpoint between the last rib and the iliac crest using a flexible measuring tape.
Skinfolds will be assessed according to the methodology established by the International
Society for the Advancement of Kinanthropometry (ISAK)35. Body composition data, including
fat mass and lean body mass, will be collected through bioelectrical impedance analysis (BIA).
Body mass index (BMI) will also be calculated by dividing weight by height squared. Lastly, for
the evaluation of the arm muscle area (AMA), we will use the formula suggested by Frisancho36.
Task 3.2 - Analysis of Blood Biomarkers
For the evaluation of lipid profile (triglycerides (TG), total cholesterol (CHOL), high-density
lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL)), glycated hemoglobin
(HbA1c), and C-reactive protein (PCR), the COBAS b 101 system, a portable device that allows
for the assessment of various analytical parameters, will be used. For this study, three capillary
blood samples will be collected via a finger prick using a single-use lancet. The assessment of
these parameters will be relevant to establish possible associations with changes in participants'
dietary habits.

Task 3.3 - Quantitative muscle component and the qualitative and muscular
performance
For the evaluation of the qualitative component and muscle performance, the FRAIL Scale and
SARC-F tools will be used to assess frailty and sarcopenia, respectively. These tools have been
validated for the Portuguese population37.
Task 3.4 - Adherence to the dietary regimen
To evaluate adherence to the nutritional intervention in the study, participants will be asked to
keep a three-day non-consecutive food diary (two weekdays and one weekend day) before and
after the intervention. Throughout the interim consultations, the 24-hour recall questionnaire
will also be used for continuous assessment of adherence to nutritional therapy.
Task 3.5 - Evaluation of quality of life
To determine health-related quality of life, the Medical Outcomes Study 36-item Short-Form
Health Survey (MOS-SF-36) questionnaire will be used38. The SF-36 consists of 36 items that
cover eight dimensions of health study and detect both positive and negative aspects. This
instrument is considered a generic measure of health as it aims to measure health concepts that
represent basic human values relevant to the functionality and well-being of each individual.
This instrument has been translated, adapted, and validated for the Portuguese population (SF-
36 v2).
Task 3.6 - Evaluation of hand grip strength
The maximum hand grip strength will be measured using the Lafayette Digital and
Dynamometer 5030D1 manual dynamometer in the dominant limb. The participant will be
seated with the elbow flexed at 90º, the shoulder flexed at 0º, and the wrist in a neutral position.
Three grip measurements will be taken with 30 seconds of rest in between, and the best of the
three measurements will be recorded.
Task 3.7 - Evaluation of cardiovascular risk level
For the evaluation of cardiovascular risk level, the SCORE2 or SCORE2 O.P38,39 algorithm
will be used39,40. The SCORE2 algorithm is applied to individuals between the ages of 40 and 69
and is based on risk factors associated with CVD such as European region risk level, gender,
age, systolic blood pressure, non-HDL cholesterol, and smoking habits. A risk percentage is
assigned between <2.5% and >25%, with stratification into low, moderate, and high risk
categories, with the specific percentages varying by risk region39. For inclusion in the present
study, individuals with a risk ≥5% (moderate and high risk) will be considered. The SCORE2

O.P. algorithm is similar to the previous one but has been adapted and validated for individuals
between the ages of 70 and 89. In this algorithm, the risk percentages range from <10%
to >40%, with the distribution among low, moderate, and high risk categories varying by risk
region38. For inclusion in the present study, individuals with a risk ≥7% (moderate and high
risk) will be considered.
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Statistical Analysis Plan
The analysis will be carried out using the Statistical Package for the Social Sciences (SPSS),
version 27.0. All normally distributed numerical variables will be presented in the format of
mean ± standard deviation. The normality of the variables will be tested using the Kolmogorov-
Smirnov test. Group differences will be determined using paired t-tests. ANOVA with repeated
measures will be used to detect changes between and within groups. Results will be considered
statistically significant at p<0.05.

Informed Consent Form (ICF)
Consent informed, clarified and free for clinical investigation
in accordance with the Helsinki Declaration and the Oviedo Convention
Study Title:
Impact
of
a
Mediterranean
Diet
(GREENHEARTEDIET
-
RATE)
on
cardiovascular disease risk factors: a randomized clinical trial.
Background: This study will be conducted under the Algarve Active Ageing - Cardiac and
Osteoarthritis Rehabilitation (A3-COR): personalized physical exercise protocol in the
rehabilitation after acute myocardial infarction or at risk of cardiovascular disease and knee
osteoarthritis, a multicenter clinical intervention study.
Study Explanation: This is a non-pharmacological intervention clinical study in which the
following will be evaluated: 1) Sociodemographic data and medical history with a close-ended
questionnaire to characterize the participant (sex, race, nationality, marital status, cohabitation,
socio-economic and educational level, current employment status, dietary habits, medication,
and medical history; 2) Body composition measurements with a bioimpedance analyzer, body
circumferences, and skinfold thickness; 3) Biochemical data including C-reactive protein level,
glycated hemoglobin, and lipid profile; 4) Health-related quality of life using the Short-Form 36
questionnaire - version 2; 5) Dietary pattern assessments using the PREDIMED questionnaire,
24-hour dietary recall, and food diary; 6) Frailty assessment using the FRAIL Scale
questionnaire; 7) Sarcopenia assessment using the SARC-F questionnaire; 8) Grip strength
measurement using a dynamometer.
Participants will be allocated to one of the following groups: nutritional intervention group
(DIET+EXERCISE) and control groups (NO DIET+EXERCISE; NO DIET+NO EXERCISE).
There will be two assessment points for all participants: at the beginning of the study and at the
end (12 weeks after the intervention). Both control groups will receive a single educational meal
session, while the intervention group will receive monthly nutritional follow-up with the
prescription of the nutritional intervention (RATE) being studied.
The nutritional component of the study, guided by a nutritionist, aims to evaluate the effect of a
nutritional intervention on improving important cardiovascular disease risk factors such as body
weight, percentage of body fat, and waist circumference. This intervention will consist of
nutritional recommendations for a healthy diet, including the predominance of plant-based
foods over the consumption of animal-based and processed foods. No food groups included in

the food pyramid will be excluded.
This assessment does not pose any risks to the health and physical and mental integrity of the
participants. The collection of blood samples through finger-prick may be considered a possible,
and only, discomfort for the participants.
Funding: This study will be funded by the A3COR Project - Algarve Active Ageing-Cardiac
and Osteoarthritis Rehabilitation (Operation Code: ALG-01-0145-FEDER-072590), financed by
the Algarve Regional Operational Program - CRESC ALGARVE 2020, and by the School of
Biotechnology of the Catholic University of Portugal. This study does not include the payment
of travel expenses or compensation to the participants.
Voluntary participation: You have the freedom to refuse to participate in this study or
withdraw your consent, suspending participation at any time. Participation is voluntary and your
refusal to participate will not involve any penalty or loss of benefits.
Confidentiality and anonymity: The collected data will be confidential and for the exclusive
use of this study. The data will be anonymized, and its processing will comply with the Data
Protection Regulation, with no publication of data that allows identification. All collected
information will be deleted immediately after the completion of the study and publication of the
respective scientific article. The collection, encoding, processing, storage, and destruction of
participant data are the responsibility of the research team. All contacts are made in a private
environment.
The researcher responsible for the study is Ana Rita Mendes Barbosa, a registered nutritionist
with the Portuguese Order of Nutritionists, with professional license number 4946N. In case of
doubts, you can contact through ana.rita.mendes.barbosa@gmail.com.
Name
of
investigator
requesting
consent:
____________________________________________
Signature:______________________________________________________ Date:__/__/_____
I declare that I have read and understood this document, as well as the verbal information provided to me by the
person who signs above. I have been assured the possibility to refuse participation in this study at any time without
any consequences. Therefore, I agree to participate in this study and allow the use of the data voluntarily provided by
me, trusting that they will only be used for this research and any potential secondary studies, and in the
confidentiality and anonymity guarantees given to me by the researcher.
Name:_________________________________________________________________
Signature:___________________________________________________ Date: __/__/_____