Cardiovascular Risk Prevention With a Mediterranean Dietary Pattern Reduced in Saturated Fat (CADIMED): Randomized Controlled Dietary Intervention Study With Metabolomic and Gut Microbiome Analyses
Phase: NA
Conditions: LDL Cholesterol
Sponsor: Universidad de Granada

 
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PROTOCOL 
 
Cardiovascular Risk Prevention With a Mediterranean Dietary 
Pattern Reduced in Saturated Fat (CADIMED):  
Randomized Controlled Intervention Study with Metabolomic 
Analysis and Gut Microbiota 
 
 
 
Version 
Date 
1.0 
11 November 2022 
1.1 
3 February 2023 
 

 
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PRINCIPAL INVESTIGATOR  
Dr. Carmen M Piernas Sánchez 
 
 
  
Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, Institute of 
Nutrition and Food Technology, Biomedical Research Centre, University of Granada. 
 
 
 
 
 
 
  
 
CLINICAL TEAM (Virgen de las Nieves University Hospital, Granada) 
Dr. Carmen Tenorio Jiménez 
Medical Dr (Specialist in Endocrinology and Nutrition) 
 
Dr. Martin Lopez de la Torre 
Medical Dr (Head of the Endocrinology and Nutrition Service) 
 
Dr. Ignacio Merino de Haro 
Primary care Dr (La Chana Health Centre, Andalusian Health Service) 
 
NURSING TEAM (Andalusian Health Service) 
Oscar Segura Calvo, Nurse 
Antonio Lebrón Arana, Nurse 
Ana Maria Rodriguez Cuesta, Nurse  
Concepción Fernandez Moreno, Nurse 
 
RESEARCH TEAM 
Dr. Concepción Aguilera García 
Professor, Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, 
Institute of Nutrition and Food Technology, Center for Biomedical Research, University of 
Granada. 
 
Dr. Carolina Gómez Llorente 
Professor, Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, 
Institute of Nutrition and Food Technology, Center for Biomedical Research, University of 
Granada.  
  
Dr. Oscar Rangel Huerta 
 
Researcher, Norwegian Veterinary Institute (Veterinærinstituttet), Oslo (Norway) 
 
Dr. Nerys Astbury 
Associate Professor, Nuffield Department of Primary Care Health Sciences, University of 
Oxford (UK) 
 
Dr. Aurora Pérez Cornago 
Associate Professor, Nuffield Department of Population Health, University of Oxford (UK) 
 
Dr. Marta Guasch Ferre 
Associate Professor, Department of Nutrition, Harvard TH Chan School of Public Health; 
and instructor in Medicine, Channing Division of Network Medicine, Harvard Medical School, 
Boston (Mass, EEUU). 
 
Dr. Lourdes Chávez Alfaro 
Medical Dr (General Medicine) and PhD student in Nutrition and Food Sciences, University 
of Granada 

 
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1. SUMMARY 
Cardiovascular disease (CVD) is the leading cause of death worldwide. The most important 
cardiovascular risk factors are modifiable, and it has been shown that an adequate diet is 
essential for the prevention of CVD due to its effect on blood pressure, lipids, obesity, 
inflammation and endothelial function. Among the main dietary macronutrients, saturated 
fatty acids (SFA) are of interest for their effects on the elevation of low-density lipoprotein 
cholesterol (LDL-C) and total cholesterol, which are associated with increased 
cardiovascular risk due to endothelial dysfunction and atherosclerosis. However, recent 
research suggests that not all SFA exerts the same effects and that certain foods such as 
red and processed meats contain a more unfavourable SFA profile, which can have 
important repercussions in relation to cardiovascular risk. The CADIMED study is an eight-
week randomized, controlled intervention study comparing the effect of an intervention that 
promotes the reduction of red and processed meat intake in a sample of 156 adult subjects 
(>18 years) and dyslipidaemia. Objectives: 1) To develop a dietary intervention that 
promotes the reduction of consumption of SFA from red and processed meats in patients 
with elevated LDL-C; 2) To determine the efficacy of this dietary intervention aimed at 
eliminating red and processed meat, in the context of a Mediterranean dietary pattern, on 
LDL-C and palmitic acid after eight weeks of intervention (main objective); 3) To determine 
the metabolic alterations parallel to the modifications in the intake of SFA from red and 
processed meat, using a complete lipid and metabolomic profile and other markers of 
cardiovascular risk (biomarkers of inflammation, endothelial damage, insulin resistance and 
diabetes; changes in blood pressure; anthropometry and C-reactive protein); 4) To assess 
the effect of the intervention on dietary intake of various nutrients and other lifestyle factors; 
5) To evaluate the effect of the intervention on metabolites derived from the gut microbiota;  
6) Establish a working panel with patient and public participation (PPP) as well as health 
professionals, to improve research management and dissemination of project results.  
Keywords: cardiovascular disease, Mediterranean diet, saturated fats, LDL cholesterol, red 
meat, palmitic acid. 
 

 
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2. BACKGROUND 
Worldwide, cardiovascular disease (CVD) is the leading cause of mortality; the World Health 
Organization estimates that, in 2019, 32% of deaths worldwide were caused by CVD, mainly 
acute myocardial infarction.1 The most important cardiovascular risk factors are modifiable 
and are associated with poor diet, lack of physical activity, tobacco and alcohol use. Among 
them, it has been shown that an adequate diet is essential for the prevention of CVD given 
its effects on blood pressure, lipids, obesity, inflammation and endothelial function.2 Of the 
dietary macronutrients, saturated fatty acids (SFA) are of great interest due to their effects 
on the elevation of low-density lipoprotein cholesterol (LDL-C) and total cholesterol, which 
are associated with increased cardiovascular risk due to endothelial dysfunction and 
atherosclerosis.3 Because of this, recommendations from different organizations and 
institutions, such as the American Heart Association and the European Society of 
Cardiology, indicate that AGS should represent less than 10% of total energy intake,4 
preferably between 5% and 6%, to prevent CVD.5  A meta-analysis of clinical trials by the 
Cochrane group has shown that reducing SFA consumption significantly decreases LDL-C 
and up to 17% cardiovascular events.6  
However, current clinical practice guidelines on the prevention and treatment of CVD 
propose a healthy diet generally low in SFA but without offering detailed or specific advice 
for effective intervention.4,7 This is important as different food sources of SFA could pose 
different health risks.8 Foods contain complex mixtures of SFA and evidence suggests that 
some of them, such as lauric, myristic, and palmitic acid, may pose different risks.9,10 Red 
meat contains a large proportion of palmitic acid [long-chain SFA (16:0)] and, although the 
current evidence is inconsistent and based mostly on observational studies, an increased 
risk of CVD associated with a higher intake of red and processed meat, compared to plant 
protein sources, has been documented, probably mediated by changes in the lipid profile.  
inflammatory or insulin resistance.11,12 However, other SFA-rich foods, such as milk and 
cheese, contain large proportions of shorter-chain [lauric (12:0) and myristic (14:0)] fatty 
acids and have not been shown to be associated with CVD.13 It has been suggested that 
dairy products have other elements such as calcium, which could counteract the 
unfavourable physiological effects of SFA.14 
The principal investigator of this project has recently led a preliminary intervention study for 
the reduction of dietary saturated fat in a population with cardiovascular risk.19 The final 
results found a non-significant decrease in SFA intake and beneficial changes in blood lipid 
profile were observed among those participants who reduced their consumption of red and 
processed meats the most, compared to those who reduced other sources of SFA such as 
dairy.19  Ultimately, the scant current evidence suggests that different sources of SFA and 
individual proportions of SFA in red and processed meat may influence cardiovascular 
profile differently, although robust intervention studies are still needed to confirm this. 
Likewise, changes in the fatty acid and blood lipid profile resulting from a dietary approach 
based on a Mediterranean diet pattern along with the elimination of red and processed meat 
consumption are largely unknown. Therefore, it is important to investigate how these dietary 
modifications affect cardiovascular risk factors in the context of a Mediterranean diet in which 

 
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the consumption of unsaturated fats from olive oil or nuts is positively associated with less 
CVD.15  
On the other hand, it is worth mentioning that despite recent efforts in public health, the 
reduction of SFA consumption through education or information programs in the population 
has not been sufficient. Previous research establishes that it is possible to reduce the intake 
of products rich in SFA by providing foods that replace them, and/or through intensive and 
adapted dietary advice, with the participation of specialized personnel and behavioural 
support.15-18 However, there is little evidence on the effectiveness of other less intensive and 
low-cost interventions that can reach a larger number of people and have a significant effect 
on the population. Simple, food-specific dietary guidance (e.g., red meat) related to 
cardiovascular risk may have greater potential to be easily disseminated and adopted by a 
large number of primary care patients who need help to improve the quality of their diets 
and thus decrease their cardiovascular risk. 
Based on what has been described, the CADIMED study aims to develop and test the 
efficacy of a dietary intervention to reduce the consumption of SFA from red and processed 
meat and to evaluate the effects on various biomarkers of cardiovascular risk in adults with 
dyslipidaemia. The determination of the lipid profile, metabolomic profile and the 
composition of the intestinal microbiota will allow a better understanding of the questions 
raised and will help to establish simple dietary guidelines based on a better approach for the 
treatment of dyslipidaemia and the prevention of CVD, relevant to the third Sustainable 
Development Goal (SDG) of the United Nations. The low cost and scalability of the 
intervention can help the Spanish health system, specifically primary care, particularly by 
helping the management of patients at risk of CVD, reducing the affected population and 
healthcare costs. In addition, the decrease in the consumption of red and processed meat 
also implies effects on global warming and environmental sustainability, contributing to 
achieving the 12th SDG. 
 

 
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3. HYPOTHESIS 
A dietary intervention based on the exclusion of red and processed meat within a 
Mediterranean dietary pattern will have a better effect on lipid profile and cardiovascular risk 
markers compared to the usual recommendation to follow a healthier lifestyle. 
 
4. OBJECTIVES 
4.1. General objective: 
To develop and determine the efficacy of a dietary intervention that promotes the elimination 
of SFA consumption from red and processed meats and to evaluate its impact on lipid and 
cardiovascular risk markers in patients with dyslipidaemia.  
 
4.2. Specific objectives: 
a. To develop the necessary elements for the application of a dietary intervention that 
promotes the reduction of the consumption of SFA from red and processed meats in 
patients with dyslipidaemia 
b. To test the efficacy of a dietary approach aimed at avoiding red and processed meat, 
in the context of a Mediterranean dietary pattern, on LDL-C and palmitic acid (main 
results) after eight weeks of intervention, compared to the usual approach of 
healthier lifestyle recommendations.  
c. To determine the metabolic alterations parallel to the changes in SFA intake, from 
red and processed meat, by means of a complete lipid and metabolomic profile, as 
well as the determination of other intermediate markers of CVD. 
d. To assess the effect of the intervention on dietary intake and other lifestyle factors. 
e. To evaluate the effect of the intervention on the composition of the gut microbiota 
and its derived plasma metabolites that could be influenced given the expected 
metabolic benefits; 
f. Establish a working panel with patient and public participation (PPP) as well as health 
professionals, to improve research management and dissemination of project 
results.  
 
 
 

 
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5. METHODOLOGY  
5.1. Experimental design 
The CADIMED study is a randomized, controlled (two parallel arms) dietary intervention 
study, where the effect of an eight-week behavioural intervention consisting of a 
Mediterranean dietary pattern with elimination of red and processed meat will be evaluated, 
compared to the general advice for decreasing SFA intake in patients with elevated LDL-C. 
To this end, 156 patients ≥18 years of age with dyslipidaemia and who express their desire 
to improve their diet and reduce the risk of CVD) will be recruited. The study will be registered 
in ClinicalTrials.gov and a Good Clinical Practice and Trial Quality Assurance strategy will 
be established (see section 11 for other ethical and legal considerations).   
 
5.2. Study population 
Inclusion criteria:  
1) Signed informed consent; 
2) Age ≥18 years to ≤75 years; 
3) Indicated to start lifestyle intervention based on recent values (e.g., last 3-6 months) of 
LDL-C≥116 mg/dL and <190 mg/dL; as long as pharmacological intervention has not been 
advised and started (ESC/EAS Guideline, Rev Esp Cardiol. 2020); 
4) Device access (e.g. smartphone, tablet) with internet and digital skills for the use of the 
mobile application;  
5) Motivation and willingness to be assigned to any of the groups to improve their diet, as 
well as commitment to carry out the assigned protocol. 
Exclusion criteria:  
1) Patients with familial hypercholesterolemia and/or on treatment with lipid-lowering drugs 
(including statins, ezetimibe, PCSK9 inhibitors, fibrates, bile acid chelators, Omega 3 fatty 
acids, nicotinic acid/Vit B3), dietary supplements or functional foods for the treatment of 
dyslipidaemia (plant sterols, monacolin, red rice yeast, fibre supplements 3-10g (plantago), 
policosanol,  berberine and soy protein/lecithin) in the last 3 months;  
2) Cardiovascular risk that makes the use of drugs necessary, for example, in patients with 
recent and/or established CVD; type 1 and 2 diabetes; 
3) Uncompensated thyroid function disorders; relevant comorbidities (including liver failure 
and cholestasis, chronic kidney disease, Cushing's syndrome, nephrotic syndrome, obesity 
grade III – BMI>40kg/m2); cancer; psychiatric disorders and/or that in the opinion of the 
researcher makes it difficult to comply with or follow up on the study; 
4) Excessive alcohol consumption: >40 g/d (4 UBEs/day) in men and >20-25 g/d (2-2.5 
UBEs/day) in women (Ministry of Health, July 2020) 

 
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4) Pregnancy, lactation, perimenopause (amenorrhea <1 year), and women who change, 
start, or end hormonal contraceptive treatment during the study;  
5) Participation in other research or dietary pattern (e.g. vegetarian and vegan) that could 
interfere with the demands of the study;  
6) Failure to provide informed consent or failure to complete the initial assessment.  
 
5.3. Procedure and data collection 
All data collected from patients will be stored in a password-protected database and 
encrypted/pseudonymized with a unique code for each patient. The code will consist of two 
letters (initials of the health centre where the patients come from) and three numbers that 
will be assigned in ascending order. Only the research team and the health authorities, who 
have a duty of confidentiality, will have access to all the data collected for the study. Only 
information that cannot be identified may be transmitted to third parties.  
1) Patient recruitment and pre-selection: Initial recruitment will be done through health 
professionals in primary or secondary care practices, who will help distribute information and 
participation in the study. Interested persons will contact the research team via telephone or 
email to confirm that they meet the inclusion requirements through a brief initial pre-selection 
questionnaire (Annex 1 – Pre-selection Questionnaire). Volunteers who meet the inclusion 
requirements will be personally scheduled for an initial visit with the research team and the 
clinical team at the Virgen de las Nieves hospital (Granada). 
 
2) Informed Consent: During the first initial visit with the research team, study information 
will be given to patients (Patient Information Sheet and Informed Consent) and the informed 
consent will be read and signed. The opportunity to resolve issues related to the study and 
participation will be offered and those who complete the informed consent will become part 
of the study.  
 
3) Questionnaires and measures: The research team will administer during the initial visit 
a general questionnaire to obtain the patient's demographic characteristics, including age, 
sex, education, socioeconomic class, alcohol intake, physical activity, diet and smoking 
(Annex 2 – Data collection notebook). The clinical team (nurse from the Virgen de las Nieves 
Hospital with links to the project) will be responsible for taking clinical data related to the 
medical history and/or use of relevant medications. Height, weight and blood pressure will 
then be measured at the beginning and at the follow-up in a standardized manner.  
In addition, help will be offered to access and use a mobile application (designed especially 
for the study) to start collecting data on diet. The app will include each patient's unique code 
and a built-in diet questionnaire that will need to be completed at least twice a week (ideally 
every day), during the first and last week of the intervention to obtain valid measures of usual 
intake. In addition, a questionnaire on the frequency of food consumption during the previous 
4 weeks will be completed to assess the eating pattern. 

 
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4) Blood samples: the nurse in charge of the project will take blood samples during the 1st 
initial visit and at the end of the follow-up of each patient, with an overnight fast of 8 hours. 
The samples will be processed in the laboratory of the Virgen de las Nieves Hospital for 
subsequent analysis in the same hospital; in addition to the transfer/storage of the remaining 
samples in our laboratory of the Institute of Nutrition and Food Technology (INYTA) (details 
on page 11-12). 
 
5) Stool samples: stool samples will be taken at the beginning and end of follow-up for 
which patients will be provided with the necessary material and instructions for collection 
(e.g. taking antibiotics) and storage of the samples. The samples will be delivered to the 
research team during the 2nd initial visit for processing and storage in our laboratory at the 
Institute of Nutrition and Food Technology (INYTA). 
 
6) Randomization and assignment of the intervention group. During the 2nd initial visit, 
and once it has been confirmed that patients meet all the inclusion requirements, the 
intervention group will be randomly assigned and the corresponding explanation to the 
patient will be carried out.    
 
Table 1. Data collection at CADIMED study visits 
 
 
Evaluation 
Preview 
1st visit 
initial 
2nd visit 
initial 
Visit 
End 
a. Pre-selection questionnaire (via telephone)  
x 
 
 
 
b. Signed consent 
 
x 
 
 
c. General questionnaire (demographics, 
lifestyle) 
 
x 
 
x 
d. Anthropometric measurements, blood 
pressure, clinical data 
 
x 
 
x 
e. Physical activity questionnaires, 24-hour 
food and diet patterns 
 
x 
x 
x 
f. Blood sample collection 
 
x 
 
x 
g. Stool sample collection 
 
 
x 
x 
h. Randomization and Intervention Allocation 
 
 
x 
 
 
5.4. Intervention  
Participants will be randomly assigned, in a 1:1 ratio, to one of two groups:  
Intervention group: Mediterranean diet without red and/or processed meat 
They will receive motivation and behavioural support through the use of the mobile 
application, which will provide them with reminders to self-monitor their changes in eating; 
while the rest of the clinical care will be usual. After randomization of the patients, the 
research team will give them more information about the use of the mobile app, with specific 

 
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indications to avoid red and processed meat in the context of a healthy Mediterranean 
dietary pattern, taking into account the key elements proposed in the dietary guidelines for 
CVD prevention. The app will provide details of Mediterranean diet recommendations, as 
well as recipes, tips and alternatives to replace meat when cooking at home or eating out. 
Participants will record their daily diet through the mobile application, during the first and last 
week of the intervention. The rest of the clinical care will be usual. 
 
Control group: Mediterranean Diet 
Similarly, the control group will be helped to download and use the mobile application, 
although they will only have access to a restricted version of it, by which they will receive 
brief and general information to maintain a healthy lifestyle following the general guidelines 
on the Mediterranean diet (without specific recommendations to reduce red and processed 
meat). Participants will also record their daily diet through the app, during the first and last 
week of the intervention. The rest of the clinical care will be usual. 
 
5.5. Randomization  
Randomization will be computer-generated, with sex, age, obesity, and diet (fiber intake) as 
stratification variables using random blocks of size 2 and 4. The computer program will 
reveal the group assigned at the same time to the researcher, without the option of viewing 
the groups that will be assigned to future patients.  
It will not be possible to blind patients to the intervention group due to the nature of the 
intervention; however, the primary outcomes of efficacy measures will be objectively 
measured (blood lipids) and are not subject to interpretation by investigators. To the extent 
possible, the study team performing all measurements will remain blind to group assignment 
during the study. 
 
5.6. Definition of variables: main and secondary results 
With the determinations and measurements derived from the CADIMED study, it is proposed 
to investigate the following results (comparing the intervention group and the control group):  
1) Main results: changes in LDL-C and fatty acid profile in erythrocytes after 8 weeks 
of intervention  
2) Secondary outcomes: 
a. Changes in other metabolites derived from lipid and metabolomic profile 
analysis, including circulating fatty acids, HDL-C, total cholesterol, 
triglycerides, and endogenous and exogenous metabolites related to 
cardiovascular risk and meat consumption (e.g., TMAO, etc.); 
b. Changes in other related markers of cardiovascular risk, including biomarkers 
of inflammation and endothelial damage, insulin resistance, and diabetes 
(glucose, insulin, HbA1c, C-reactive protein, IL6, TNF-alpha), blood pressure, 
weight, and body and waist mass index; 

 
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c. Changes in the gut microbiota and derived metabolites 
d. Changes in dietary intake (measured by 24-h recall): SFA intake, total energy 
intake, total fat, sugar, fiber, salt, general dietary patterns, red and processed 
meat consumption. 
e. Changes in other lifestyle-related factors: collected through physical activity, 
smoking, and alcohol questionnaires 
 
3) Evaluation of the process: analyses of both quantitative and qualitative measures 
will be included to understand whether the intervention has worked and through 
which processes. These measures include: the rate of recruitment of volunteers and 
the rate of patients completing the study; acceptance of the intervention through 
questionnaires at the end of follow-up; the use of the mobile application to carry out 
changes in the diet as well as the self-monitoring of intake; measures that evidence 
the contamination of the control group; changes in patients' ability and knowledge 
about diet, SFA, and cardiovascular disease.  
 
5.7. Data analysis 
Sample size 
A recent randomized, controlled, peer-to-population trial testing similar dietary approaches 
to reduce SFA intake showed a long-term change in LDL-C of approximately 15 mg/dL 
(SD26).19 If 78 participants are recruited in each group (n = 156), it is expected to detect a 
minimum difference of 15 mg/dl between the intervention group and the control, with a power 
or detection capacity of 90%, an error α = 0.05 and a dropout rate of 20% using intention-
to-treat analysis,  so the final sample size includes an additional 20% of participants to 
mitigate dropouts.  
Statistical analysis of primary and secondary outcomes 
Baseline characteristics will be summarized in descriptive statistics in each study group, 
statistical analysis will be performed with STATA IC 16.0 software (College Station, TX: 
StataCorp LLC 2019). Descriptive statistics will be presented with standard deviation (SD) 
and 95% confidence intervals. Continuous variables will be presented as mean and standard 
deviation unless the use of the median and interquartile range is necessary; Categorical 
variables will be expressed as frequencies and percentages.  
Efficacy results will be analysed by intent to treat, i.e., all participants will be assessed 
according to the group to which they are randomly assigned, regardless of their adherence. 
The analysis of effectiveness measures will use data from all participants for whom a 
baseline and monitoring measure exists. A linear regression model will be used to calculate 
the difference in measures with 95% confidence intervals, with adjustment of the initial 
values of the dependent variable and the place of recruitment. Before the analysis, the 
assumptions of linear regression, specifically homoscedasticity and the normality of the 
residual distribution, will be explored. Where these assumptions are not met, appropriate 
non-parametric analyses shall be used. The sensitivity of the results to confounders due to 

 
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differences in baseline characteristics will also be examined. A second sensitivity analysis 
will use the techniques of "baseline observation carried forwards" (using the initial measure 
of the variable) and "multiple imputation" (using information about the assigned group and 
the characteristics of the participants) to impute missing values in the variables of the main 
outcome. An exploratory analysis of subgroups of results will be carried out according to 
sociodemographic characteristics.  
 
5.8. Determination of metabolomic profiles and gut microbiota 
This project will employ a metabolic and metagenomic profiling approach (gut microbiota) 
using state-of-the-art analytical technologies to provide a deeper understanding of metabolic 
responses to dietary intervention (reduction of red/processed meat intake) in the context of 
a Mediterranean dietary pattern. Therefore, lipid profiling and a complete metabolomic and 
metagenomic analysis will be performed to investigate specific changes caused by the 
intervention compared to the control. The determinations will be made at the hospital 
(general biochemistry) as well as at the Institute of Nutrition and Food Technology (INYTA). 
Once the main determinations explained below have been carried out and with prior 
consent, the surplus samples with their corresponding code will be stored at -80ºC in the 
Biobank of the Public Health System of Andalusia (BBSSPA), for future analyses related to 
the project, including aliquots with leukocytes for DNA extraction and erythrocytes for studies 
of antioxidant defense enzymes.   
 
5.8.1. Lipid metabolism, endothelial function and inflammatory markers 
Fasting venipuncture blood samples will be obtained at the beginning and end of follow-up: 
two 10 mL tubes of K2E EDTA, and two 10 mL tubes of serum separator gel. First, part of 
the sample will be processed in the hospital according to its own established protocols to 
obtain blood biochemistries for the blood lipid fractions: total cholesterol, LDL-C (primary 
outcome), HDL-C, non-HDLc, triglycerides, glucose, insulin, HbA1c and CRP. 
The rest of the samples will be processed at INYTA to obtain plasma and stored (-80 °C) 
until they are processed for analysis. The new LC-MS will be used to determine a specific 
plasma fatty acid profile, including palmitic acid (primary outcome), among others. Other 
plasma biomarkers of endothelial function, such as ICAM-1, VCAM-1, ELAM-1, and MPO, 
will be determined using Luminex®'s xMAP technology and  MULTIPLEX kits; as well as 
endothelin-1 and angiotensin II using  ELISA kits. Inflammatory biomarkers in plasma will 
also be determined with Luminex® xMAP and  HSTCMAG-28SK kits, including IL-6, IL-
8/CXCL8, IL-10, IL-23, TNF-α, IFN-γ. ELISA kits will be used to quantify lipopolysaccharides 
(LPS) and LPS-binding lipoprotein (LBP). 
 
5.8.2. Metabolomic determinations 
Plasma metabolomic profiles will be determined in a non-targeted manner using an LC-MS 
platform, although targeted analyses will also be proposed to determine endogenous and 

 
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exogenous metabolites specifically related to cardiovascular risk and meat consumption 
(e.g., TMAO, etc.). Non-targeted approaches are used to simultaneously measure 
hundreds/thousands of metabolic traits that then allow a metabolic signature to be profiled. 
For metabolite extraction, plasma samples will be mixed with an organic solvent preparation 
for protein precipitation, cooled, centrifuged, and filtered before being analysed. During this 
step, quality control (QC) samples will be prepared by taking 10 μL aliquots from all samples.  
Additional CC samples will be collected from subgroups of each group for further 
fragmentation analysis. The analysis will be carried out using two different LC methods 
coupled to an Orbitrap Q-Exactive instrument operating in positive and negative ionization 
modes, respectively, in order to relatively separate and quantify plasma metabolites. Pre-
processing of the raw data generated will include removal of non-significant information, 
alignment, filtering, QC titration, drift correction, and normalization of the intensity of each 
ion, and will be carried out using MD-DIAL, XCMS, or a combination of proprietary R-based 
codes.  A final table will be obtained containing: 1) mass/load ratio (m/z, mass error < 3 
ppm); 2) retention time (RT, min) and 3) chromatographic peak area for each of the 
metabolites. Additional fragmentation data will be acquired to improve information on 
metabolites. From current public metabolomic databases and from our own collection, 
metabolites will be mapped based on their m/z signal and fragmentation data. Identification 
will be carried out using appropriate chemical standards where possible.  The information 
will be reported following the method suggested by Summer et al.  
 
 
5.8.3. Determination of the composition of the gut microbiota 
It will be carried out by 16S rRNA analysis of the samples obtained and previously processed 
and stored at -80º C. DNA will be obtained from the stool sample using the QIAamp DNA 
stoolmimi kit (Qiagen, Barcelona, Spain) and the PureLink™ Microbiome DNA purification 
Kit (Invitrogen). The quality and quantity of the same will be checked spectrophotometrically 
(NanoDrop ND-100). The variable V3-V4 region of the 16S rRNA gene will be amplified 
using specific oligonucleotides, by PCR. A second amplification will then be performed using 
barcode oligonucleotides. The purified PCR product will be sequenced on illumina's MiSeq 
platform. 
 
5.8.4. Statistical analysis of metabolomic and metagenomic data 
A "Knowledge Discovery from Databases" (DCBD) process will be followed, which will 
consist of: 
- Selection of variables of interest and groups of subjects for analysis. 
- Identify distinct clinical feature sets that are grouped into particular individual groups 
(phenotypic sets) without regard to their microbiota/metabolomic background. 
- Examine the response associated with the intervention in each individual identified 
phenotypic set. 

 
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- Identify metabolomic networks composed of DMRs and metabolites that interact with each 
other. 
- Integration of all previously identified patterns into a multi-layered space using genetic 
algorithms. The idea of this final step is, once the interesting variables derived from each of 
the previous approaches (phenotype, microbiota and metabolomics) have been identified, 
to incorporate them all together for the construction of a predictive model that allows the 
classification of good and bad responders to treatment. 
To this end, different statistical methodologies will be used to identify anomalies, groups or 
patterns of metabolites, including principal component analysis (PCA) for the initial 
clustering. To differentiate metabolite patterns between the intervention and control groups, 
before and after the intervention, a novel multivariate approach, orthogonal partial least 
squares multiblock analysis of variance (AMOPLS), will be used. The AMOPLS consists of 
the application of a core-based multiblock OPLS approach on the pure matrices and those 
augmented by the residues arising from the ANOVA decomposition of the design response 
matrix and provides, as a product, a global view of the important effects on the model, which 
allows its interpretation. Analysis of variance and/or linear regression will test the main 
effects of: 1) time (follow-up vs. baseline); 2) intervention group vs. control; 3) interaction 
time x intervention group. Univariate analyses will be used to compare baseline means 
between the intervention and control groups using Student's t-tests. A false discovery rate 
(q) will be calculated to reduce false positives with a cut-off point < 0.1.  
For the extraction of bacterial DNA from faeces, the commercial QIAamp Stool Mini Kit 
(Qiagen, Venlo, The Netherlands) will be used. The quality and quantity of the extracted 
DNA will be spectrophotometrically checked (NanoDrop ND-100). The variable region V4 
515F-806R of the 16S rRNA gene will be amplified using specific s-oligonucleotides, by 
PCR. A second amplification will then be performed using barcode oligonucleotides. The 
purified PCR product will be sequenced on illumina's MiSeq platform, and the protocols to 
be followed will be those recommended by the manufacturer. Data analysis. The analysis 
will be carried out with the QUIIME2 v.2019.4 program. The data will be filtered with the q2-
dada2plugin implemented in the R software (DADA2 method), obtaining the corresponding 
ASV (Amplicon sequence variance) correspondents. The taxonomic assignment will be 
determined with respect to the GreenGenes database (v.13.8). Both alpha diversity 
(Shannon index) and beta diversity (Unifrac index) will be determined. For the detection of 
differences in taxonomic composition, the ANCOM method implemented in QUIIME2 will be 
used. A prediction of the metabolic functions of the metagenomes will also be made using 
the PICRUSt2 tool. 
For all these analyses  , statistical software such as SIMCA® (Umetrics, Umea, Sweden), 
Stata and R will be used. Artificial intelligence methods, such as ARM (association rule 
mining) and GA (generic algorithms) will be used to combine and analyze the phenotypic 
and metabolomic data in order to understand the most predictive patterns that are 
associated with better outcomes in this population.  
 
 

 
15 
 
6. USEFULNESS AND APPLICABILITY 
Patients at risk of cardiovascular disease will benefit the most from the results of this project. 
Clinical guidelines for reducing cardiovascular risk emphasize the importance of dietary 
change and recommend that all patients with dyslipidaemia be supported to change their 
diet. However, dietary interventions are uncommon and there is limited evidence of their 
effectiveness in the setting of routine primary or secondary care. At present, there are no 
comprehensive resources to enable physicians and health care professionals to offer brief 
dietary advice on the management of dyslipidaemia. This project will develop and test a 
dietary intervention that is easy to implement by clinical staff without much prior knowledge, 
but also intuitive so that patients with elevated LDL cholesterol can follow and maintain a 
healthier diet. If we get the expected results, this project will provide an effective approach 
to offer dietary advice within the Spanish healthcare system. The low cost and increased 
scalability of this intervention could substantially help the Spanish healthcare system to 
manage the large number of patients at risk of CVD who could benefit from this intervention. 
This in turn could translate into lower costs derived from medical care in the Spanish 
population. 
In addition, the results of this project will inform food-based dietary guidelines on the most 
promising approach to the treatment of dyslipidaemia at the population level. Therefore, the 
findings of this study will be relevant to other countries where red and processed meat intake 
is above dietary recommendations, e.g. the UK or the US. The intervention materials and 
mobile app that will be developed in this project (potential intellectual property) will provide 
a low-cost, widely disseminated approach to providing dietary advice to all people who need 
it.  
Finally, if a reduction in red and processed meat leads to health benefits, this will also have 
implications for global warming and environmental sustainability, as consuming less red 
meat could help protect the environment as well as improve human health. Therefore, this 
project will also contribute to achieving the Sustainable Development Goals (Sustainable 
Development Goals) by promoting healthy eating habits that are compatible with human and 
planetary health. 
 
7. WORK PLAN 
The CADIMED project is divided into four phases of work with a total duration of three years 
and two blocks aimed at the communication and dissemination of the results, as well as 
project management, which will happen simultaneously throughout the three years (see 
Table 2). The first year will be dedicated to the development of the intervention, recruitment 
of the working panel with health professionals and patients/interested persons, development 
of study materials and protocols along with the ethical approval of the intervention study. 
Approximately 15 months will be spent on participant recruitment and intervention. The last 
year will be devoted to metabolomic determinations. The communication and dissemination 
of results has been planned towards the end of the second year and during the third year. 
The management activities of the project will be regular (monthly, quarterly or annual) to 
ensure its development and the achievement of results and indicators. 

 
16 
 
The PI will be responsible for the coordination and administration of the project and will be 
supported by the rest of the research team and other collaborators. Steering committee 
meetings will be held every 6 months to provide progress on the project.  
In addition, a research assistant will be hired to assist with patient recruitment and sample 
and data collection, as well as to assist with the overall management of the project. 
Specifically, the research assistant will support the PI in the development of the dietary 
intervention, including the materials that will be distributed to participants as part of the 
intervention; mobile application development; recruitment of PPIs and health professionals 
in the study; contact and management of participants; baseline and follow-up 
measurements; determinations in biological samples; Collaboration in data management 
and data analysis. The PI and the research group will have the capacity to train this person 
in the required skills and competencies. 
 
Table 2. CADIMED Study Timeline 
ACTIVITY 
YEAR 1 
YEAR 2 
YEAR 3 
MONTH 
Phase I – Development of the intervention 
 
 
 
 
 
 
 
 
 
 
 
 
 
Preparation of materials for the intervention 
 
 
 
 
 
 
 
 
 
 
 
 
1-6 
Phase II – Working Panel 
 
 
 
 
 
 
 
 
 
 
 
 
 
Recruitment of health professionals 
 
 
 
 
 
 
 
 
 
 
 
 
1-12 
PPP Recruitment 
 
 
 
 
 
 
 
 
 
 
 
 
1-12 
Phase III – CADIMED Randomized, Controlled Intervention Study 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study protocols, consent forms, ethical approval 
 
 
 
 
 
 
 
 
 
 
 
 
1-9 
Recruitment of participants, implementation of the intervention, data collection 
 
 
 
 
 
 
 
 
 
 
 
 
9Mon-24 
Trial data management and statistical analysis 
 
 
 
 
 
 
 
 
 
 
 
 
21-30 
Phase IV – Lipid, Metabolomic, and Metagenomic Profiling  
 
 
 
 
 
 
 
 
 
 
 
 
 
Lipid profile and other CVR markers 
 
 
 
 
 
 
 
 
 
 
 
 
21-33 
Metabolomic and metagenomic profiles  
 
 
 
 
 
 
 
 
 
 
 
 
 
Data management and statistical analysis 
 
 
 
 
 
 
 
 
 
 
 
 
21-33 
Communication, dissemination and exploitation of data 
 
 
 
 
 
 
 
 
 
 
 
 
 
Communication, dissemination and exploitation plan 
 
 
 
 
 
 
 
 
 
 
 
 
21 
Participation in outreach and engagement activities 
 
 
 
 
 
 
 
 
 
 
 
 
18, 36 
Manuscript preparation and submission 
 
 
 
 
 
 
 
 
 
 
 
 
24, 36 
Preparation of results and attendance at congresses 
 
 
 
 
 
 
 
 
 
 
 
 
24, 36 
Preparation of materials for use in the health system 
 
 
 
 
 
 
 
 
 
 
 
 
27 
Project Management 
 
 
 
 
 
 
 
 
 
 
 
 
 
Meetings with team members 
 
 
 
 
 
 
 
 
 
 
 
 
Monthly 
Data Management Plan Development 
 
 
 
 
 
 
 
 
 
 
 
 
9 
Administrative meetings and project reports 
 
 
 
 
 
 
 
 
 
 
 
 
6, 18, 30 
Trial steering committee meetings, coordination and evaluation seminars 
 
 
 
 
 
 
 
 
 
 
 
 
6, 18, 33 
 
 
 

 
17 
 
8. DISSEMINATION PLAN 
1) Academic results 
- This project will produce high-quality, open-access publications, including potential journals 
such as Circulation, Annals of Internal Medicine, PloS Medicine or BMC Medicine, which 
are the main journals with a recent interest in these topics ranging from basic to clinical 
metabolic research. At least 2 other important publications are expected to emerge from this 
project: one describing the results of all metabolomics evaluations; and another with dietary 
and other CVD-related outcomes. 
- The PI and research team have a proven track record in this field internationally (UK, USA), 
with frequent invitations to national and international conferences. 
2) Public Health  
- A guide with recommendations will be developed using the results of this project and will 
use the established links to promote knowledge transfer within the public health and regional 
health system (e.g. Health Ministry) as well as in Hospital Endocrinology Units and in 
Primary Care. 
3) Media  
- The results are expected to be disseminated in news published by the National Societies 
of Endocrinology and Nutrition (e.g. SEEDO, SEEN, SEÑ). 
- National and regional health-related television programs. 
- Dissemination of research activities in the communication unit of the University of Granada, 
including science fairs, or related activities in museums. 
- Dissemination on social networks, including Researchgate, Facebook and Twitter. 
- International coverage: the research team has extensive international collaboration 
networks, with multiple projects and publications in collaboration with international 
researchers. 
4) Participants and members of the public (PPI) 
- Diverse PPI members will be actively involved by writing summaries of the results and 
assisting with the communication strategy. PPI members will also participate in the 
preparation of infographics that will be publicly available in the Endocrinology Units of the 
Hospitals and in Primary Care. 
 
 
 
 
 
 

 
18 
 
9. BUDGET 
For the realization of this project, funds are contemplated from two calls:  
1. Call Ministry of Science and Innovation, Knowledge Generation Projects 2021, PID2021-
127312OA-I00 (see table 3 with budget granted). This budget will be used to cover most of 
the expenses derived from the execution of the project, with the exception of the analysis of 
the microbiota.  
2. Ramon y Cajal 2020 Call, RYC2020-028818-I, Ministry of Science and Innovation. It has 
a budget of €42,000 for the execution of research activities, which will be used to pay for 
microbiota analyses.  
 
Table 3. Budget granted from the Ministry of Science and Innovation Call 
Knowledge Generation Projects 2021 PID2021-127312OA-I00 
Budget granted 
GOODS AND SERVICES 
BUDGET (€) 
Material Fungible:  
 
Blood determinations: plasma lipid profiles, biochemistry, biomarkers of 
CVD and inflammation.  
Sample of 156 patients x 2 points= 352 x €52/each=€18300 
18300 
Metabolomic Determinations  
Sample of 156 patients x 2 points= 352 x 100€/each=35200€ 
35200 
Other materials: printing, letters, creation and maintenance of databases 
2000 
Software Licenses:  
 
Mobile App Software 
5000 
EXPENSES OF HIRING PERSONNEL SPECIFICALLY FOR THE 
PROJECT including salaries, employer Social Security contributions, 
legally established indemnities and other expenses derived from the hiring 
duly justified 
 
Research support staff: 
 
Research Assistant (e.g. Undergraduate Student in Nutrition)  
64500 
TRAVEL, ACCOMMODATION AND MEALS:  
 
Travel and subsistence allowances for International Congresses 
 
FENS (Federation of European Nutrition Societies). 3 dias (Europa)  
1300 
OTHER EXPENSES  
 
Open Access main results 
2500 
Auditing 
1200 
 
 
DIRECT COSTS  
130,000 
INDIRECT COSTS (21%) 
27,300 
TOTAL GRANTED  
157,300 

 
19 
 
10. INFRASTRUCTURE AND EQUIPMENT 
Both the University of Granada and the Institute of Nutrition and Food Technology (INYTA), 
in the Department of Biochemistry and Molecular Biology II, as well as the collaboration with 
the Virgen de las Nieves hospital (Dr. Carmen Tenorio), make the project have all the 
necessary structural and technological resources to guarantee the adequate development 
of this research project and meet the needs of those who work in this institution. The link 
with the Virgen de las Nieves clinical team will facilitate access to the participants of the 
CADIMED study, as well as the necessary resources to obtain medical records, samples 
and blood biomarkers. In addition, the department has invested in the technical resources 
necessary to carry out the metabolomic analyses, including a new LC-MS, with high analysis 
speed and sensitivity, obtained with funds from the Ministry of Science, Innovation and 
Universities with ERDF funding (exp. No. EQC2019-006110-P). Currently, the department 
has a very advanced information system, with high-speed internet, a data centre equipped 
with state-of-the-art servers, a data storage area based on fibre optics, automatic backup 
systems and uninterrupted power supply. These advanced technological structures are 
complemented by a wide range of office, statistical, technological and document 
management software, as well as open access to any publication through the open access 
library.  
 
Research Team  
Dr. Carmen Piernas, PI of this project, has eight years of experience in postdoctoral research 
and holds a PhD in Nutrition and Epidemiology from the University of North Carolina at 
Chapel Hill (USA). The PI of this project has worked in the Health Behaviours team  in the 
Department of Primary Care and Health Sciences of the University of Oxford (UK), so she 
brings important experience and skills in nutrition, epidemiology and behavioural sciences, 
as well as has demonstrated a strong leadership capacity to carry out projects successfully,  
from the conception of the research idea, to the application for funding and the conduct of 
the research. It was the PI of the Primary Care Shopping Intervention for Cardiovascular 
Disease Prevention (PCSHOP) study that was conducted at the University of Oxford; this 
study aimed to test new behavioural approaches to achieve reductions in SFA intake among 
patients with elevated LDL-C and has provided fundamental preliminary results for its 
advancement in this area of research and finding effective dietary approaches in CVD 
prevention. The project has resulted in several relevant publications, the main one in PLOS 
Medicine. During the management of the PCSHOP study, Dr. Piernas gained important skills 
and knowledge to successfully plan and conduct randomized clinical trials; he also led more 
recent behavioural dietary interventions (SwapSHOP and SaltSwap studies) in which he 
participated in the development of two mobile apps that help give dietary advice and facilitate 
self-management of dietary changes.  
Dr. Piernas joins the CTS-461 group "Biochemistry of Nutrition, Therapeutic Implications" 
(www.bionit.ugr.es), co-directed by Dr. Concepción Aguilera within the Department of 
Biochemistry and Molecular Biology II and the Institute of Nutrition and Food Technology 
(INYTA), located at the Biomedical Research Centre of the University of Granada (UGR) 

 
20 
 
and the Biosanitary Institute of Granada (IBS. Granada) accredited by the ISCIII. This group 
is an excellent environment for the successful implementation of the CADIMED project. Prof. 
Aguilera and her group have demonstrated extensive experience in the fields of omics, in 
particular genetics, epigenetics and metabolomics. Prof. Concepción Aguilera and her team 
members are leading highly competitive research on the molecular basis of obesity and 
metabolic syndrome, and are one of the most fruitful institutions in medical sciences at the 
University of Granada, for which they have obtained funding for several projects, including 
nutritional interventions, within the biomedical sciences:  
1. 
Association between genetic variants, biomarkers of oxidative stress, inflammation 
and cardiovascular risk in children with obesity. GENOBOX study. 2012/2015. Carlos III 
Health Institute – Ministry of Science and Innovation (EU funding): €143,330 
2. 
Puberty and metabolic risk in obese children: epigenetic alterations and 
pathophysiological and diagnostic implications (PUBMEP). 2017/2020. Carlos III Health 
Institute – Ministry of Science and Innovation (EU funding): €131,890 
3. 
Omics and artificial intelligence as tools to understand the molecular mechanisms of 
insulin resistance in children with obesity during puberty (IBEROMIC). 2021/2023. Carlos 
III Health Institute – Ministry of Science and Innovation (EU funding): €173,030 
4. 
Clinical evaluation of an olive oil enriched with bioactive compounds with 
antihypertensive and cardioprotective properties. Research and development of new 
products for a healthier shopping basket that improves child nutrition (MEDKIDS). CIEN 
(CDTI – Ministry of Industry and Energy). 2019/2023. Total funding: €247,026 
5. 
Evaluation of an olive oil spread, enriched with phytosterols and bioactive 
compounds with cholesterol-lowering and cardioprotective properties. Research and 
development of new products for a healthier shopping basket that improves child nutrition 
(MEDKIDS). CIEN (CDTI – Ministry of Industry and Energy). 2019/2023. Total funding: 
140,000 € 
 
This project has also formed a new multidisciplinary research group that has a wide range 
of experts, from nutritional sciences to behavioural sciences and biochemistry/molecular 
biology, as well as clinical experts in endocrinology and nutrition. This team will cover the 
knowledge, skills and competencies necessary to ensure that the project is viable, credible 
and obtains the proposed results: 
Dr. Carmen Tenorio Jiménez, Medical Specialist in Endocrinology and Nutrition (Virgen de 
las Nieves University Hospital). His group has relevant experience in dietary interventions 
in patients with risk factors for CVD and obesity. Lead and coordinate the clinical team to 
recruit more health care professionals to help perform the intervention. 
 
Dr. Martín López de la Torre, Medical Practitioner, Head of the Endocrinology and Nutrition 
Service (Virgen de las Nieves University Hospital). His group has relevant experience in 
dietary interventions in patients with related risk factors. She will support Dr. Tenorio in 
coordinating the clinical team. 
 

 
21 
 
Dr. Carolina Gómez Llorente, Associate Professor of the Department of Biochemistry and 
Molecular Biology II, Faculty of Pharmacy, Institute of Nutrition and Food Technology, 
Biomedical Research Center, University of Granada. He has relevant experience in the 
study of the gut microbiota as well as the integration of related clinical data.  
 
Dr. Oscar Rangel Huerta, Norwegian Veterinary Institute (Veterinærinstituttet), Oslo, 
Norway. His expertise is related to metabolomic determinations as well as statistical 
analyses of metabolomic data. 
 
Dr. Nerys Astbury, Nuffield Department of Primary Care Health Sciences, University of 
Oxford, UK. She is a senior nutrition researcher with expertise in behavioral science and 
dietary interventions in primary care and other clinical settings, involving patients with 
cardiovascular disease, diabetes, and obesity. 
 
Dr. Aurora Perez Cornago, Nuffield Department of Population Health, University of Oxford, 
United Kingdom. She is a senior researcher (university research professor) with a 
background in nutrition and epidemiology. He has researched the relationship between meat 
consumption and cardiovascular diseases and has worked on large clinical trials for the 
prevention of cardiovascular diseases, such as PREDIMED. 
 
Dr. Marta Guasch Ferre, researcher, Department of Nutrition, Harvard T.H. Chan School 
of Public Health; Instructor of Medicine, Channing Division of Network Medicine, Harvard 
Medical School, Boston (Mass, USA). She has a background in nutrition, with experience in 
clinical trials and epidemiological studies around diet and chronic non-communicable 
diseases, including cardiovascular diseases. 
 
Dr. Lourdes Chávez Alfaro, Medical Practitioner (General Medicine), PhD student in 
Nutrition and Food Sciences, University of Granada. He has relevant training and 
experience in clinical studies, as well as in the development and application of interventions 
to improve habits in at-risk patients. They will be in charge of carrying out the recruitment of 
patients, development and application of the intervention, laboratory analysis and 
preparation of samples for analysis, preparation and updating of the database, statistical 
analysis and will participate in the dissemination and preparation of the results publications. 
 
 
 
 
 
 
 
 
 
 
 

 
22 
 
 11. ETHICAL AND LEGAL CONSIDERATIONS 
The development of the project will be carried out in compliance with the Declaration of 
Helsinki of the World Medical Association 1964 and ratifications of the following assemblies 
(Tokyo 75, Venice 83, Hong Kong 89, Somerset West 96, Scotland 00, Seoul 08 and 
Fortress 13) on ethical principles for medical research on human beings, RD 1090/2015, of 
24 December.  of clinical trials, specifically the provisions of Article 38 on Good Clinical 
Practice, and the Convention on Human Rights and Biomedicine), signed in Oviedo on 4 
April 1997 and successive updates. 
The investigators participating in this study undertake that all clinical data collected from the 
study subjects will be separated from the personally identifiable data in order to ensure the 
anonymity of the patients; respecting Organic Law 3/2018, of 5 December, on the Protection 
of Personal Data and guarantee of digital rights, Law 41/2002, of 14 November (basic 
regulation of patient autonomy and rights and obligations in terms of clinical information and 
documentation), as well as Law 3/2001, of 28 May, (regulating informed consent and 
patients' medical records),  Law 3/2005, of 7 March, amending Law 3/2001 and Decree 
29/2009 of 5 February, which regulates access to electronic medical records. 
All data related to the project will be coded/pseudonymized, protecting the patient's identity, 
specifically for this project. Only the research team and the health authorities, who have a 
duty of confidentiality, will have access to all the data collected for the study. Only 
information that cannot be identified may be transmitted to third parties, maintaining the 
encryption/pseudonymization. Once the study is completed, the data will be pseudonymised 
for future use (always related to this line of research), with express permission for this in the 
informed consent of the participants. 
The handling of samples will follow the provisions of Law 14/2007 of 3 July on Biomedical 
Research, in RD 1301/2006, of 10 November, which establishes the quality and safety 
standards for the donation, procurement, evaluation, processing, preservation, storage and 
distribution of human cells and tissues and approves the coordination and operation rules 
for their use in humans and in the RD 1716/2011, of 18 November, which establishes the 
basic requirements for the authorisation and operation of biobanks for the purposes of 
biomedical research and the treatment of biological samples of human origin, and regulates 
the operation and organisation of the National Register of Biobanks for biomedical research. 
The samples will be processed in a coded way, protecting the patient's identity. Once the 
study is completed, the samples will be stored coded in the Biobank of the Public Health 
System of Andalusia (BBSSPA), with prior signed consent, for future analyses related to the 
project.   
The processing, communication and transfer of data will be carried out in accordance with 
the provisions of the General Data Protection Regulation (Regulation (EU) 2016/679 of the 
European Parliament and of the Council, of 27 April 2016) and Organic Law 3/2018, of 5 
December, on the Protection of Personal Data and Guarantee of Digital Rights.  
The data collected will only be used for the purposes of the research study described in the 
protocol and kept for the time necessary to achieve the objectives of the study and in 
accordance with applicable legislation. 

 
23 
 
Personal data will be processed exclusively for the research work referred to in this 
document. The processing of the data of this study is legitimized by the signed consent to 
participate. No data will be transferred to third parties unless legally obliged. The patient 
may exercise their rights of access, rectification, deletion and portability of their data, 
limitation and opposition to their processing, in accordance with the provisions of the 
General Data Protection Regulation (GDPR 2016/679) before the principal investigator of 
the project, and may obtain information in this regard by contacting the research and clinical 
team.  
The processing of personal data will be carried out using techniques to maintain anonymity 
through the use of codes, so that personal identity is completely hidden during the 
investigation process.  
Based on the results of the research work, scientific communications may be prepared to 
be presented at congresses or scientific journals, but they will always be done with grouped 
data and nothing that can identify the patient will ever be disclosed. 
 
12. BIBLIOGRAPHY 
1. World Health Organization. Cardiovascular diseases (CVDs). WHO: Copenhague; 2021. 
Disponible en: https://www.who.int/news-room/fact-sheets/detail/cardiovascular-diseases-
(cvds)  
2. Verschuren WMM, Boer JMA, Temme EHM. Optimal diet for cardiovascular and planetary 
health. Heart 2022;108:1234-9. 
3. Davignon J, Ganz P. Role of endothelial dysfunction in atherosclerosis. Circulation 
2004;109:27-32. 
4. Visseren FLJ, Mach F, Smulders YM, et al. ESC 2021 Guidelines on the Prevention of 
Cardiovascular Disease in Clinical Practice. Rev Esp Cardiol 2022;75:364-910. 
5. American Heart Association. Saturated Fat. AHA: Texas: 2021. Disponible en: 
https://www.heart.org/en/healthy-living/healthy-eating/eat-smart/fats/saturated-fats  
6. Hooper L, Martin N, Jimoh OF, et al. Reduction in saturated fat intake for cardiovascular 
disease. Cochrane Database Syst Rev 2020; 8:CD011737. 
7. Pavia AA, Alcocer MA, Ruiz ED, et al. Mexican clinical practice guidelines for the 
diagnosis and treatment of dyslipidemias and atherosclerotic cardiovascular disease. Arch 
Cardiol Mex 2022;92:1-62. 
8. Astrup A, Magkos F, Bier DM, et al. Satured fats and health: a reassessment and proposal 
for food-based recommendations: JACC State-of-the-Art review. J Am Coll Cardiol 
2020:76:844-57. 
9. Chowdhury R, Warnakula S, Kunutsor S, et al. Association of dietary, circulating, and 
supplement fatty acids with coronary risk: a systematic review and meta-analysis. 2014 
160:398-406.  

 
24 
 
10. Zong G, Li Y, Wanders AJ, et al. Intake of individual saturated fatty acids and risk of 
coronary heart disease in US men and women: two prospective longitudinal cohort studies. 
BMJ 2016; 355:i5796.  
11. Guasch M, Satija A, Blondin SA, et al. Meta-analysis of randomized controlled trials of 
red meat consumption in comparison with various comparison diets on cardiovascular risk 
factors. Circulation 2019;139:1828-45.  
12. Key TJ, Appleby PN, Bradbury KE, et al. Consumption of meat, fish, dairy products, and 
eggs and risk of ischemic heart disease. Circulation 2019;139:2835-45.  
13. Visioli F, Poli A. Fatty acids and cardiovascular risk. Evidence, lack of evidence, and 
diligence. Nutrients 2020;12:3782. 
14. de Oliveira Otto MC, Mozaffarian D, Kromhout D, et al, Dietary intake of saturated fat 
food source and incident cardiovascular disease: the Multi-Ethnic Study of Atherosclerosis. 
Am J Clin Nutr 2012;96:397-404.  
15. Estruch R, Ros E, Salas J, et al. Primary prevention of cardiovascular disease with a 
Mediterranean diet supplemented with extra-virgin olive oil or nuts. N Engl J Med 2018; 
378:e34. 
16. Reidlinger DP, Darzi J, Hall WL, et al. How effective are current dietary guidelines for 
cardiovascular disease prevention in healthy middle-aged and older men and women? A 
randomized controlled trial. Am J Clin Nutr 2015;101:922-30.  
17. Vafeiadou K, Weech M, Altowaijri H, et al. Replacement of saturated with unsaturated 
fats had no impact on vascular function but beneficial effects on lipid biomarkers, E-selectin, 
and blood pressure: results from the randomized, controlled Dietary Intervention and 
VAScular function (DIVAS) study. Am J Clin Nutr 2015;102:40-8.  
18. Lin JS, O'Connor E, Whitlock EP, et al. Behavioral counseling to promote physical 
activity and a healthful diet to prevent cardiovascular disease in adults: a systematic review 
for the U.S. Preventive Services Task Force. Ann Intern Med 2010;153:736-50. 
19.  Piernas C, Aveyard P, Lee C, et al. Evaluation of an intervention to provide brief support 
and personalized feedback on food shopping to reduce saturated fat intake (PC-SHOP): a 
randomized controlled trial. PLoS Med 2020; 17:e1003385.

 
25 
 
ANNEX 1. PRE-SELECTION QUESTIONNAIRE 
 
The participant:  
Yes No 
You have read and signed your informed consent 
 
 
You are 18 years of age or older but less than 76 years of age 
 
 
Has recent LDL-C values of 116 to 189 mg/dL with no indication/initiation of 
pharmacological intervention 
 
 
Is motivated to participate in the study and make changes to their diet 
 
 
You have access to a smartphone mobile phone  with internet and digital 
skills for the use of the mobile application 
 
 
Are participating in another study or are following an incompatible diet 
(vegetarians, vegans) 
 
 
Are pregnant or have pregnancy plans during the time allotted for the study 
 
 
Is breastfeeding 
 
 
Is in perimenopause (amenorrhea < 1 year) 
 
 
Started, stopped, or made changes to hormonal contraceptive/replacement 
therapy dosing (last 3 months) or plan changes in the next two months 
 
 
You are taking or need lipid-lowering medications/supplements (see Table 1)   
 
Have a history of cardiovascular, thyroid, renal, hepatic, psychiatric disease, 
familial hypercholesterolemia, type 1 or type 2 diabetes (see Table 2) 
 
 
It has a BMI ≥ 40 kg/m2 
 
 
Are male and consume > 40 g/day (4 UBEs/day) of alcohol or are female 
and consume > 25 g/day (2.5 UBEs/day) 
 
 
 
Table 1. LIPID-LOWERING MEDICATIONS/SUPPLEMENTS (used within the last 3 
months) 
 
Yes 
No 
Statins (atorvastatin, rosuvastatin, simvastatin...) 
 
 
Ezetimiba 
 
 
Inhibidores de la PCSK9 (evolocumab, alirocumab, bococizumab…) 
 
 
Fibratos (fenofibrate, gemfibrozilo, clofibrate...) 
 
 
Bile acid chelators (cholestyramine, colestipol hydrochloride...) 
 
 
Omega-3 fatty acids 
 
 
Nicotinic acid/niacin/vitamin B3 
 
 
Plant sterols/phytosterols 
 
 
Monacoline 
 
 
Red Yeast Rice 
 
 
Fiber supplements, 3 to 10 g (Plantagus psyllium)  
 
 
Policosanol 
 
 
Berberine 
 
 
Soy protein/lecithin 
 
 
Hormonal contraceptives 
 
 
 
Table 2. MEDICAL HISTORY 
 
Yes No 
Kidney disease (chronic kidney disease, nephrotic syndrome...) 
 
 
Liver disease (liver failure, cholestasis...) 
 
 
Thyroid disease 
 
 

 
26 
 
Cardiovascular disease (myocardial infarction, cerebral vascular event, heart 
failure, arrhythmia, angina...) 
 
 
Systemic arterial hypertension 
 
 
Type 1 diabetes 
 
 
Type 2 diabetes 
 
 
Cushing's Syndrome 
 
 
Familial hypercholesterolemia 
 
 
Mental health disturbance 
 
 
Autoimmune disease 
 
 
Cancer 
 
 
Perimenopause (amenorrhea < 1 year) 
 
 
Current breastfeeding 
 
 
Current pregnancy 
 
 

 
27 
 
ANNEX 2. DATA COLLECTION NOTEBOOK 
 
A. GENERAL DATA QUESTIONNAIRE 
 
Age: ___ years 
 
Gender:  Men 
 Women 
 
Employment status: What is your current employment status, or last occupation? 
 
Active worker (incl. leave of absence) 
☐ 
Temporary incapacity for work 
☐ 
Unemployed (available and looking for work) 
☐ 
Student 
☐ 
Permanent incapacity for work 
☐ 
Dedicated to household chores 
☐ 
Performing social work or charitable activities without pay 
☐ 
I've never had a paid job 
☐ 
Other 
☐ 
 
Educational Level: What is your maximum level of completed studies? 
 
Cannot read or write 
☐ 
Incomplete primary schools: they can read and write without having finished primary 
education 
☐ 
Completed Primary: Complete LOGSE Primary or five approved EGB courses 
☐ 
First stage of Secondary Education: School Graduate, Elementary Baccalaureate, EGB or 
ESO completed 
☐ 
Baccalaureate studies: upper baccalaureate, BUP, baccalaureate new plan, PREU or COU 
☐ 
Intermediate vocational training: industrial official, FPI, intermediate level training cycles 
☐ 
Higher level vocational training: industrial master's degree, FPII, higher level training 
cycles 
☐ 
First-cycle university studies: university diploma, architecture and technical engineering 
☐ 
Second-cycle university studies: bachelor's, bachelor's, architecture and engineering 
☐ 
University studies of doctorate, postgraduate, master's, MIR or equivalent 
☐ 
Another possibility.  
☐ 
 
Economic situation: Can you tell which of the following ranges best represents your 
entire household's net monthly income, after deductions for taxes, Social Security, etc.? 
 
Less than 570 euros   
☐ 
From 570 to less than 800 euros   
☐ 
From 800 to less than 1050 euros   
☐ 
From 1050 to less than 1,300 euros   
☐ 
From 1,300 to less than 1,550 euros  
☐ 
From 1,550 to less than 1,800 euros  
☐ 
From 1,800 to less than 2,200 euros   
☐ 
From 2,200 to less than 2,700 euros   
☐ 
From 2,700 to less than 3,600 euros   
☐ 
From 3,600 to less than 4,500 euros   
☐ 

 
28 
 
From 4,500 to less than 6,000 euros  
☐ 
From 6,000 euros onwards   
☐ 
He doesn't know   
☐ 
No answer  
☐ 
 
Ethnicity or cultural group: To which ethnicity or cultural group do you belong? 
 
White – Caucasian 
☐ 
Asian 
☐ 
Maghreb 
☐ 
Amerindian 
☐ 
Gypsy 
☐ 
Black 
☐ 
Other 
☐ 
He doesn't know 
☐ 
No answer 
☐ 
 
Tobacco use:  
 
Smoker 
☐ 
     Number of cigarettes/tobacco per day:  
 
Ex-smoker 
☐ 
Non-smoker 
☐ 
He doesn't know 
☐ 
No answer 
☐ 
 
Alcohol Consumption: How often do you drink alcohol? 
 
4 to 7 times a week (Number of unidades__________) 
☐ 
2 to 3 times a week (Number of unidades__________) 
☐ 
2 to 4 times a month (Number of unidades__________) 
☐ 
1 or fewer times per month (Number of unidades__________) 
☐ 
Never 
☐ 
He doesn't know 
☐ 
No answer 
☐ 
 
Health-related quality of life: In general, you would say that your health is:  
 
Excellent or very good 
☐ 
Good 
☐ 
Regular 
☐ 
Bad 
☐ 
He doesn't know 
☐ 
No answer 
☐ 
 
 
 
Family history: select those diseases that your parents, grandparents, children and/or 
siblings have: 

 
29 
 
 
High blood pressure (high blood pressure) 
☐ 
Obesity 
☐ 
Diabetes (blood sugar) 
☐ 
Myocardial infarction 
☐ 
Cerebral vascular disease 
☐ 
Hypercholesterolemia (high cholesterol) 
☐ 
Elevated triglycerides 
☐ 
 
 
 
 
 
 
 
 
B. PHYSICAL EXAMINATION AND CLINICAL DATA 
 
Initial assessment 
Final Evaluation 
Date 
 
 
Weight (kg) 
 
 
Size (cm) 
 
 
BMI (kg/m2) 
 
 
Waist circumference (cm) 
 
 
Blood pressure (mmHg) 
PAS1: ____ 
PAS2: ____ 
PAS3: ____ 
PAD1: ____ 
PAD2: ____ 
PAD3: ____ 
PAS1: ____ 
PAS2: ____ 
PAS3: ____ 
PAD1: ____ 
PAD2: ____ 
PAD3: ____ 
Heart rate (bpm) 
 
 
Relevant medical history 
 
 
Use of relevant medications 
 
 
 
C. LABORATORY DATA 
 
Initial assessment 
Final Evaluation 
Date 
 
 
Total cholesterol 
 
 
c-LDL 
 
 
c-HDL 
 
 
Triglycerides 
 
 
Glucose 
 
 
Insulin 
 
 
HbA1c 
 
 
PCR 
 
 
 
 
 
 
 
 
 
 
D. BIOMARKERS OF ENDOTHELIAL FUNCTION, INFLAMMATORIES AND 
COMPOSITION OF THE GUT MICROBIOTA 
 
Initial assessment 
Final Evaluation 
Date 
 
 
ICAM-1 
 
 

 
30 
 
VCAM-1 
 
 
ELAM-1 
 
 
MPO 
 
 
Endothelin-1 
 
 
Angiotensin II 
 
 
IL-6 
 
 
IL-8/CXCL8 
 
 
IL-10 
 
 
IL-23 
 
 
TNF-α 
 
 
IFN-γ 
 
 
LPS 
 
 
LBP 
 
 
 

 
31 
 
E. QUESTIONNAIRE ON PHYSICAL ACTIVITY AND SEDENTARY LIFESTYLE 
The following questions are related to how long you do different types of physical 
activity. Try to answer, even if you don't consider yourself an active person. 
At work 
Think first about the time you spend at work, whether it is paid or unpaid employment, 
studying, maintaining your home, or the main activity you do daily. In these questions, 
"intense physical activities" refer to those that involve significant physical exertion and 
cause a great acceleration in breathing or heart rate. On the other hand, "moderate-
intensity physical activities" are those that involve moderate physical exertion and cause 
a slight acceleration in breathing or heart rate. 
Q1. Does your job require intense physical activity  that involves significant acceleration 
of breathing or heart rate (such as lifting, digging, or construction work) for at least 10 
minutes at a time? 
 Do not → go to P4. 
 Yes 
Q2. How many days a week? ____ days a week 
 
Q3. How long each day? ____ minutes each day 
Q4. Does your job require moderate-intensity activity  that involves a slight acceleration 
of breathing or heart rate (such as brisk walking or carrying light weights) for at least 10 
minutes at a time? 
 Do not → go to P7. 
 Yes 
Q5. How many days a week? ____ days a week 
 
Q6. How long each day? ____ minutes each day 
To get around 
We will leave aside physical activities at work to know how you move from one place to 
another. For example, how they go to school, shopping, the market, work, etc. 
Q7. Do you walk or bike for at least 10 consecutive minutes on your commute? 
 Do not → go to P10. 

 
32 
 
 Yes 
Q8. How many days a week? ____ days a week 
 
Q9. How long each day? ____ minutes each day 
In your free time 
The following questions exclude physical activity at work and for commuting. Now we 
will ask about other physical activities that you practice in your free time. 
Q10. In your free time, do you play strenuous sports  that involve significant acceleration 
of breathing or heart rate (such as running, playing soccer) for at least 10 minutes at a 
time? 
 Do not → go to P13. 
 Yes 
Q11. How many days a week? ____ days a week 
 
Q12. How long each day? ____ minutes each day 
P13. In your free time, do you engage in any moderate-intensity activity  that involves a 
slight acceleration of breathing or heart rate (such as brisk walking, cycling, swimming, 
playing volleyball) for at least 10 minutes at a time? 
 Do not → go to P16. 
 Yes 
P14. How many days a week? ____ days a week 
 
P15. How long each day? ____ minutes each day 
Sedentary behavior 
The next question refers to the time you usually spend sitting or lying down at work, at 
home, on the commute or with your friends. It includes time spent at a work table, sitting 
with friends, traveling by bus or train, playing cards, watching TV, using a mobile phone 
or video games, but it does not include time spent sleeping.  
P16. How much time do you typically spend sitting or lying down on a typical day? 
 ___ hours : ___ minutes 

 
33 
 
F. FEEDING QUESTIONNAIRE  
1. Do you use olive oil as your main cooking fat? 
 Yes 
 No 
2. How much olive oil do you consume in total per day? 
(including the one used for frying, meals out, salads, etc.) 
____ tablespoons 
3. How many servings of vegetables do you eat per day? 
(garnishes or accompaniments = 1/2 portion) 1 portion = 200 g 
____ ration 
4. How many pieces of fruit do you eat per day? 
____ Parts 
5. How many servings of red meat, hamburgers, sausages or 
sausages do you consume per day? (1 serving = 100-150 g) 
____ Servings 
6. How many servings of butter, margarine or cream do you 
consume per day? (single serving: 12 g) 
____ Servings 
7. How many carbonated and/or sugary drinks (soft drinks, colas, 
tonics, bitters) do you consume per day? 
____ Drinks 
8. Do you drink wine? How much do you consume per week? 
____ glasses per 
week 
9. How many servings of legumes do you eat per week? (1 plate 
or portion = 150 g) 
____ Servings 
10. How many servings of seafood do you eat per week? (1 dish, 
piece or portion = 100-150 g of fish, or 4-5 pieces, or 200 g of 
seafood) 
____ Servings 
11. How many times do you consume commercial (non-
homemade) pastries such as cookies, custards, sweets or cakes 
a week? 
____ times per 
week 
12. How many servings of nuts do you eat a week? (1 serving = 
30 g) 
____ Servings 
13. Do you prefer chicken, turkey or rabbit meat instead of beef, 
pork, hamburgers or sausages? (chicken meat = 1 piece or 
serving of 100-150 g) 
 Yes 
 No 
14. How many times a week do you eat cooked vegetables, 
pasta, rice, or other dishes seasoned with tomato, garlic, onion, 
or leek sauce simmered with olive oil (sofrito)? 
____ times per 
week 

 
34 
 
 
G. FOOD FREQUENCY QUESTIONNAIRE 
Please read the instructions carefully:  
 
For each food, check the box that indicates the frequency of consumption, on average, during the past month. 
You will find foods that are consumed by season, if you did not consume them during the past month, try to 
remember their frequency of consumption in that season. 
 
AVERAGE CONSUMPTION IN A MONTH 
I. DAIRY 
Never or  
less than 
once a month 
Per 
month 
Per week 
Up to date 
 
1-3 
1 
2
-
4 
5-
6 
1 
2-
3 
4-
6 
6+ 
1. 
Whole milk (1 cup, 200 cc) 
 
 
 
  
 
 
 
 
2. 
Semi-skimmed milk (1 cup, 200 cc) 
 
 
 
  
 
 
 
 
3. 
Skim milk (1 cup, 200 cc) 
 
 
 
  
 
 
 
 
4. 
Condensed milk (1 tablespoon) 
 
 
 
  
 
 
 
 
5. 
Cream or heavy cream (1/2 cup)  
 
 
 
  
 
 
 
 
6. 
Milkshakes (1 glass, 200 cc) 
 
 
 
  
 
 
 
 
7. 
Full-fat yogurt (1, 125 g) 
 
 
 
  
 
 
 
 
8. 
Yogur discremated (1, 125 g) 
 
 
 
  
 
 
 
 
9. 
Petit Suisse (1.55 g) 
 
 
 
  
 
 
 
 
10. 
Cottage cheese or curd (1/2 cup) 
 
 
 
  
 
 
 
 
11. 
Portioned or creamy cheese (1.25 g) 
 
 
 
  
 
 
 
 
12. 
Other cheeses: cured, semi-cured (Manchego, Bola, 
Emmental...) (50 g) 
 
 
 
  
 
 
 
 
13. 
White or fresh cheese (Burgos, goat...) (50 g) 
 
 
 
  
 
 
 
 
14. 
Natillas, flan, puding (1, 130 cc) 
 
 
 
  
 
 
 
 
15. 
Ice cream (1 cone) 
 
 
 
  
 
 
 
 
II. EGGS, MEAT, FISH 
(100-150g dish or serving, except where indicated) 
Never the 
less than 
once a month 
Per 
month 
Per week 
Up to date 
 
1-3 
1 
2
-
4 
5-
6 
1 
2-
3 
4-
6 
6+ 
16. 
Chicken eggs (1) 
 
 
 
  
 
 
 
 
17. 
Chicken or turkey WITH skin (1 serving or piece) 
 
 
 
  
 
 
 
 
18. 
Skinless chicken or turkey (1 serving or piece) 
 
 
 
  
 
 
 
 
19. 
Beef (1 portion) 
 
 
 
  
 
 
 
 
20. 
Pork (1 serving) 
 
 
 
  
 
 
 
 
21. 
Lamb meat (1 portion) 
 
 
 
  
 
 
 
 
22. 
Rabbit or hare (1 portion) 
 
 
 
  
 
 
 
 
23. 
Liver (beef, pork, chicken) (1 serving) 
 
 
 
  
 
 
 
 
24. 
Other organ meats (brains, heart, sweetbreads) (1 portion) 
 
 
 
  
 
 
 
 
25. 
Serrano ham or shoulder ham (1 slice, 30 g) 
 
 
 
  
 
 
 
 
26. 
York ham, cooked ham (1 slice, 30 g) 
 
 
 
  
 
 
 
 
27. 
Processed meats (salami, chorizo, black pudding, 
mortadella, sausages, sausage, sobrasada) (50 g) 
 
 
 
  
 
 
 
 
28. 
Patés, foie gras (25 g) 
 
 
 
  
 
 
 
 
29. 
Burger (1, 50 g), meatballs (3 units) 
 
 
 
  
 
 
 
 
30. 
Bacon, bacon, bacon (50 g) 
 
 
 
  
 
 
 
 
31. 
White fish: grouper, sole, sea bream, hake, whiting... (1 
dish, piece or portion) 
 
 
 
  
 
 
 
 
32. 
Oily fish: sardines, tuna, bonitl, mackerel, salmon (1 dish, 
piece or portion 130 g) 
 
 
 
  
 
 
 
 
33. 
Salted fish: cod, salted fish (1 portion, 60 g dry) 
 
 
 
  
 
 
 
 
34. 
Oysters, clams, mussels and the like ( 6 units) 
 
 
 
  
 
 
 
 
35. 
Squid, octopus, baby squid, cuttlefish (1 portion, 200 g) 
 
 
 
  
 
 
 
 

 
35 
 
36. 
Crustaceans: prawns, prawns, langoustines, etc. (4-5 
pieces, 200 g) 
 
 
 
  
 
 
 
 
37. 
Canned fish and seafood (sardines, anchovies, bonito, tuna) 
(1 small can or half a normal can, 50 g) 
 
 
 
  
 
 
 
 
38. 
Fish and seafood in oil (sardines, anchovies, bonito, tuna) (1 
small can or half a normal can, 50 g) 
 
 
 
  
 
 
 
 
III. VEGETABLES  
(200g dish or portion, except where indicated) 
Never the 
less than 
once a month 
Per 
month 
Per week 
Up to date 
 
1-3 
1 
2
-
4 
5-
6 
1 
2-
3 
4-
6 
6+ 
39. 
Swiss chard, spinach 
 
 
 
 
 
 
 
 
40. 
Cabbage, cauliflower, broccoli 
 
 
 
 
 
 
 
 
41. 
Lettuce, endives, escarole (100 g) 
 
 
 
 
 
 
 
 
42. 
Crudo tomato (1.50 g) 
 
 
 
  
 
 
 
 
43. 
Carrot, pumpkin (100 g) 
 
 
 
  
 
 
 
 
44. 
Green beans 
 
 
 
  
 
 
 
 
45. 
Aubergines, zucchini, cucumbers 
 
 
 
  
 
 
 
 
46. 
Peppers (150 g) 
 
 
 
  
 
 
 
 
47. 
Asparagus 
 
 
 
  
 
 
 
 
48. 
Andalusian gazpacho (1 glass, 200 g) 
 
 
 
  
 
 
 
 
49. 
Other vegetables (artichoke, leek, thistle, celery) 
 
 
 
  
 
 
 
 
50. 
Onion (1/2 piece, 50 g) 
 
 
 
  
 
 
 
 
51. 
Garlic (1 clove) 
 
 
 
  
 
 
 
 
52. 
Parsley, thyme, bay leaf, oregano, etc. (1 pinch) 
 
 
 
  
 
 
 
 
53. 
Commercial French fries (1 bag, 50 g) 
 
 
 
  
 
 
 
 
54. 
Homemade fries (1 portion, 150 g) 
 
 
 
  
 
 
 
 
55. 
Roasted or cooked potatoes 
 
 
 
  
 
 
 
 
56. 
Mushrooms, chanterelles, mushrooms 
 
 
 
  
 
 
 
 
IV. FRUITS (one piece or portion) 
Never the 
less than 
once a month 
Per 
month 
Per week 
Up to date 
 
1-3 
1 
2
-
4 
5-
6 
1 
2-
3 
4-
6 
6+ 
57. 
Orange (1), grapefruit (1), or tangerines (2) 
 
 
 
  
 
 
 
 
58. 
Banana (1) 
 
 
 
  
 
 
 
 
59. 
Apple or pear (1) 
 
 
 
  
 
 
 
 
60. 
Strawberries (6 units, 1 dessert plate) 
 
 
 
  
 
 
 
 
61. 
Cherries, picotas, plums (1 dessert dish) 
 
 
 
  
 
 
 
 
62. 
Peach, apricot, nectarine (1) 
 
 
 
  
 
 
 
 
63. 
Sandía (1 tajada, 200-250 g) 
 
 
 
  
 
 
 
 
64. 
Melon (1 slice, 200-250 g) 
 
 
 
  
 
 
 
 
65. 
Kiwi (1 piece, 100 g) 
 
 
 
  
 
 
 
 
66. 
Grapes (one bunch, 1 dessert plate) 
 
 
 
  
 
 
 
 
67. 
Olives (10 units) 
 
 
 
  
 
 
 
 
68. 
Fruits in syrup or juice (2 units) 
 
 
 
  
 
 
 
 
69. 
Dates, dried figs, raisins, prunes (150 g) 
 
 
 
  
 
 
 
 
70. 
Almonds, peanuts, hazelnuts, pistachios, pine nuts (30 g) 
 
 
 
  
 
 
 
 
71. 
Walnuts (30 g) 
 
 
 
  
 
 
 
 
72. 
How many days a week do you eat fruit for dessert?  
 0 
 1 
 2 
 3 
 4 
 5 
 6 
 7 
 
 
 
 
 
V. LEGUMES AND CEREALS 
(150 g plate or serving) 
Never the 
less than 
once a month 
Per 
month 
Per week 
Up to date 

 
36 
 
 
1-3 
1 
2
-
4 
5-
6 
1 
2-
3 
4-
6 
6+ 
73. 
Lentils (1 dish, 150 g cooked) 
 
 
 
  
 
 
 
 
74. 
Beans (pinted, white or black) (1 plate, 150 g cooked) 
 
 
 
  
 
 
 
 
75. 
Chickpeas (1 plate, 150 g cooked) 
 
 
 
  
 
 
 
 
76. 
Peas, broad beans (1 plate, 150 g cooked) 
 
 
 
  
 
 
 
 
77. 
White bread, sliced bread (3 slices, 75 g) 
 
 
 
  
 
 
 
 
78. 
Black or wholemeal bread (3 slices, 75 g) 
 
 
 
  
 
 
 
 
79. 
Breakfast cereals (30 g) 
 
 
 
  
 
 
 
 
80. 
Whole grains: muesli, rolled oats, all-bran (30 g) 
 
 
 
  
 
 
 
 
81. 
White rice (60 g raw) 
 
 
 
  
 
 
 
 
82. 
Pasta: noodles, macaroni, spaghetti, oysters (60 g raw) 
 
 
 
  
 
 
 
 
83. 
Pizza (1 serving, 200 g) 
 
 
 
  
 
 
 
 
VI. OILS AND FATS 
(one tablespoon or individual serving. For frying, spreading, 
dipping on bread, dressing, or salads, use in total:) 
Never the 
less than 
once a month 
Per 
month 
Per week 
Up to date 
1-3 
1 
2
-
4 
5-6 
1 
2-
3 
4-
6 
6+ 
84. 
Olive oil (1 tablespoon) 
 
 
 
  
 
 
 
 
85. 
Extra virgin olive oil (1 tablespoon) 
 
 
 
  
 
 
 
 
86. 
Pomace olive oil (1 tablespoon) 
 
 
 
  
 
 
 
 
87. 
Corn oil (1 tablespoon) 
 
 
 
  
 
 
 
 
88. 
Sunflower oil (1 tablespoon) 
 
 
 
  
 
 
 
 
89. 
Soybean oil (1 tablespoon) 
 
 
 
  
 
 
 
 
90. 
Mixture of the above (1 tablespoon) 
 
 
 
  
 
 
 
 
91. 
Margarine (single serving, 12 g) 
 
 
 
  
 
 
 
 
92. 
Butter (single serving, 12 g) 
 
 
 
  
 
 
 
 
93. 
Lard (10 g) 
 
 
 
  
 
 
 
 
94. 
Brand of olive oil you regularly use:  
 
VII. PASTRIES AND PASTRIES 
Never the 
less than 
once a month 
Per 
month 
Per week 
Up to date 
1-3 
1 
2
-
4 
5-6 
1 
2-
3 
4-
6 
6+ 
95. 
Maria biscuits (4-6 units, 50 g) 
 
 
 
  
 
 
 
 
96. 
Wholemeal or fibre biscuits (4-6 units, 50 g) 
 
 
 
  
 
 
 
 
97. 
Chocolate biscuits (4 units, 50 g) 
 
 
 
  
 
 
 
 
98. 
Homemade pastries and biscuits (50 g) 
 
 
 
  
 
 
 
 
99. 
Croissant, ensaimada, tea cakes or other commercial 
industrial pastries (1.50 g) 
 
 
 
  
 
 
 
 
100. 
Donuts (1) 
 
 
 
  
 
 
 
 
101. 
Muffins (1-2 units) 
 
 
 
  
 
 
 
 
102. 
Cakes (1, 50 g) 
 
 
 
  
 
 
 
 
103. 
Churros, porras and similar (1 portion, 100 g) 
 
 
 
  
 
 
 
 
104. 
Chocolates y bombones (30 g) 
 
 
 
  
 
 
 
 
105. 
Cocoa powder or soluble (1 teaspoon for dessert) 
 
 
 
  
 
 
 
 
106. 
Nougat (1/8 loaf, 40 g) 
 
 
 
  
 
 
 
 
107. 
Mantecados, marzipan (90 g) 
 
 
 
  
 
 
 
 
VIII. MISCELLANEOUS 
Never the 
less than 
once a month 
Per 
month 
Per week 
Up to date 
 
1-3 
1 
2
-
4 
5-
6 
1 
2-
3 
4-
6 
6+ 
108. 
Croquettes, fritters, dumplings, precooked (1) 
 
 
 
  
 
 
 
 
109. 
Soups and creams (1 plate) 
 
 
 
  
 
 
 
 
110. 
Mustard (one teaspoon for dessert) 
 
 
 
  
 
 
 
 
111. 
Commercial mayonnaise (1 tablespoon, 20 g) 
 
 
 
  
 
 
 
 
112. 
Fried tomato sauce, ketchup (1 teaspoon) 
 
 
 
  
 
 
 
 
113. 
Spicy: tabasco, pepper, paprika (1 pinch) 
 
 
 
  
 
 
 
 
114. 
Salt (1 pinch) 
 
 
 
  
 
 
 
 

 
37 
 
115. 
Jams (1 teaspoon) 
 
 
 
  
 
 
 
 
116. 
Sugar (1 teaspoon) 
 
 
 
  
 
 
 
 
117. 
Honey (1 teaspoon) 
 
 
 
  
 
 
 
 
118. 
Snacks other than chips, worms, popcorn, corn, etc. (1 bag, 
50 g) 
 
 
 
  
 
 
 
 
119. 
Other foods frequently consumed: 
 
- 
 
 
 
 
  
 
 
 
 
- 
 
 
 
 
  
 
 
 
 
- 
 
 
 
 
  
 
 
 
 
IX. BEVERAGES 
Never the 
less than 
once a month 
Per 
month 
Per week 
Up to date 
 
1-3 
1 
2
-
4 
5-
6 
1 
2-
3 
4-
6 
6+ 
120. 
Carbonated with sugar: drinks with cola, lemonades, tonics, 
etc. (1 bottle, 200 cc) 
 
 
 
  
 
 
 
 
121. 
Low-calorie carbonated drinks, light drinks (1 bottle, 200 cc) 
 
 
 
  
 
 
 
 
122. 
Fresh orange juice (1 glass, 200 cc) 
 
 
 
  
 
 
 
 
123. 
Natural juices of other fruits (1 glass, 200 cc) 
 
 
 
  
 
 
 
 
124. 
Bottled or canned fruit juices (200 cc) 
 
 
 
  
 
 
 
 
125. 
Decaffeinated coffee (1 cup, 50 cc) 
 
 
 
  
 
 
 
 
126. 
Coffee (1 cup, 50 cc) 
 
 
 
  
 
 
 
 
127. 
Tea (1 cup, 50 cc) 
 
 
 
  
 
 
 
 
128. 
Wort (100 cc) 
 
 
 
  
 
 
 
 
129. 
Rosé wine glass (100 cc) 
 
 
 
  
 
 
 
 
130. 
Muscat wine glass (50 cc) 
 
 
 
  
 
 
 
 
131. 
Young red wine glass, of the year (100 cc) 
 
 
 
  
 
 
 
 
132. 
Glass of aged red wine (100 cc) 
 
 
 
  
 
 
 
 
133. 
White wine glass (100 cc) 
 
 
 
  
 
 
 
 
134. 
Glass of cava (100 cc) 
 
 
 
  
 
 
 
 
135. 
Beer (1 jug, 330 cc) 
 
 
 
  
 
 
 
 
136. 
Liqueurs, anise or anisetae... (1 glass, 50 cc) 
 
 
 
  
 
 
 
 
137. 
Spirits: whiskey, vodka, gin, cognac (1 glass, 50 cc) 
 
 
 
  
 
 
 
 

 
38 
 
H. 24-HOUR REMINDER 
Instructions:  
 
Before you begin, please read the following observations that will help you optimize data 
collection. 
 
The objective of this survey is to know their daily consumption of food and beverages. 
Write down, as accurately as possible, all foods and drinks consumed in the last 24 
hours or the previous day. You can start with yesterday's breakfast and continue until 
you complete the memory of the entire day's diet. Write down the foods consumed 
between meals.  
 
Write down the type/variety of the food (whole or skimmed milk, white or wholemeal 
bread, type of meat, type of fish, type of oil, etc.) and estimate the amount consumed in 
household measurements or servings (large, medium, small); the information shown on 
the packaging of many foods may be useful for this purpose. Write down the oil used in 
culinary preparations, bread, sugar or drinks consumed (water, soft drink, milk, coffee, 
juice, alcoholic beverages, etc.). Also, record the culinary preparation method  (cooked, 
fried, roasted, etc.). 
 
To make it easier to remember, initially write down the menu consumed at each meal 
and then describe the ingredients in detail. To help memory, it is practical to remember 
where you ate, with whom, at what time, who prepared the meal, etc; These details will 
help you "get into the situation."  
 
Has the food you're about to record been different for any reason?  
 Yes    No 
 
If yes, please indicate why:  
 
_______________________________________________________________ 
 
Indicate if you take supplements (type and amount): 
 
 _______________________________________________________________ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
39 
 
Date corresponding to the day of remembrance: 
Breakfast 
Menu (food and preparation) 
Ingredients (type and quantity) 
 
 
 
Drinks: 
Sugar: 
Mid-morning 
Menu (food and preparation) 
 
 
Ingredients (type and quantity) 
 
Food 
Menu (food and preparation) 
 
• 
First 
 
 
• 
Second 
 
 
• 
Dessert 
 
 
Ingredients (type and quantity) 
 
 
Drinks: 
Bread: 
Oil (Type): 
Snack 
Menu (food and preparation) 
 
 
Ingredients (type and quantity) 
 
 
Dinner 
Menu (food and preparation) 
 
• 
First 
 
 
• 
Second 
 
 
• 
Dessert 
 
Ingredients (type and quantity) 
 
 
Drinks: 
Bread: 
Oil (Type): 
Between meals 
Menu (food and preparation) 
 
Ingredients (type and quantity) 
 
 
 

 
40 
 
LOG OF PROTOCOL CHANGES 
 
Version 
Date 
Changes 
1.0 
11 November 2022 
 
1.1 
3 February 2023 
1. Inclusion of the SAS nursing team 
2. Size of the randomization blocks: change from 8 
and 16 to 2 and 4 due to the improvement in the 
balance and distribution of characteristics in the 
intervention groups  
3. Inclusion of sensitivity analyses: baseline 
observation carried forwards (using the initial 
measure of the variable) and multiple imputation 
(using information about the assigned group and the 
characteristics of the participants) to impute missing 
values in the variables of the main outcome 

CADIMED Study 
Patient Information and Informed Consent 
 
Title: Prevention of Cardiovascular Risk with a Mediterranean Dietary Pattern reduced in Saturated Fat 
(CADIMED Study). 
Principal Investigator: Dr. Carmen Piernas (carmenpiernas@ugr.es); Tel 615210894 
 
 
 
Name and Surname PARTICIPANT: ........................................................................................................ 
 
Name and Surname INFORMANT: ........................................................................................................ 
 
MARK WITH A CROSS 
YES 
NO 
I have read the information document that accompanies this consent (Patient Information), 
understand the risks and benefits involved, as well as the requirements for participation in 
the study 
 
 
I have received enough information about the study, I have talked to the study staff and I 
have been able to ask questions about it 
 
 
I understand that my participation is voluntary and I am free to participate or not participate 
in the study. If you choose not to participate, this decision will not affect the medical care 
you receive 
 
 
I understand that I can withdraw from the study at any time, without explanation. In this 
case, no new data will be added to the database. 
 
 
In accordance with LO15/1999, I understand that I can exercise the rights of access, 
modification, opposition and cancellation of my data. To do this, you must contact the 
Principal Investigator of the project. You can also request the destruction of the samples. 
 
 
I have been informed that the donation/information obtained will only be used for the 
specific purposes of the study and studies related to cardiovascular disease 
 
 
I understand that the study information will be confidential and that no unauthorized person 
will have access to the data or samples. 
 
 
I give my permission to be informed of my data and other personal data that may be 
obtained in the course of the investigation, including any unexpected discoveries that may 
occur, provided that this information is necessary to avoid serious damage to my health 
 
 
I freely consent to participate in this project 
 
 
I give my consent for the surplus samples to be kept anonymously in the Biobank of the 
Andalusian Public Health System for future use in related research 
 
 
 
 
Patient's signature  
 
Name and surname .................................................................................................................................... 
Date: .................................... 
 
 
 
Signature of the reporter  
 
Name and surname: .................................................................................................................................. 
Date: ....................................