Study of the Postprandial Effect of Olive Leaf Biscuits and Tea Consumption on Plasma Antioxidant Status, as Well as on Metabolic Syndrome Indicators, in Healthy Individuals
Phase: NA
Conditions: Oxidative Stress, Inflammation Biomarkers
Sponsor: University of the Aegean

 
 
 
NUTRITIONAL INTERVENTION PROTOCOL – CLINICAL STUDY 
"Study of the Postprandial Effects of Consuming Olive Leaf Products and Tea on 
Plasma Antioxidant Status and Metabolic Syndrome Markers in Healthy 
Individuals." 
Research Team 
Scientific Supervisor: Dr. Antonios Koutelidakis, Assistant Professor at the 
Department of Food and Nutrition, University of the Aegean 
Researcher: Panagiota Potsaki, PhD Candidate at the Department of Food Science 
and Nutrition, University of the Aegean 
 
Funding Organization  
This study will take place under the project "OLIHERB." 
 
October 2024 

✓ Σύνοψη Πρωτοκόλλου 
1. Protocol Summary 
This is the protocol of a randomized controlled nutritional intervention - clinical study to 
investigate the bioactivity of preparations (biscuits and tea) with dried olive leaves. 
This is a nutritional intervention - clinical study with a cross-over design (cross over design: 
acute study). The main objective of the study is to investigate the postprandial effect of olive 
leaves on selected biomarkers and on the antioxidant status of plasma. 
The participants of the study will be healthy volunteers, aged 18-65 years, who will come 
from areas of Greece and will be based on the island of Lemnos, in order to be able to attend 
the days of the study. The study will be carried out in the specially designed area of the 
Human Nutrition Unit, University of the Aegean, in collaboration with external doctors. The 
total duration of the study will be 15 days (3 axes). 
Participants will be randomly divided into three groups. On the first day, the first group will 
consume a meal rich in carbohydrates and fats, which will consist of 2-3 biscuits made with 
butter, flour and sugar and drops of caramel coloring (E150a), along with a glass of natural 
mineral water with the addition of caramel coloring (E150a) at a refrigerator temperature of 
~4°C (control group), the second group 2-3 biscuits made with butter, flour and sugar and 
dried olive leaf powder, along with a glass of natural mineral water with the addition of 
caramel coloring (E150a) at a refrigerator temperature of ~4°C (intervention group A) and 
the third group 2-3 biscuits made with butter, flour and sugar and drops of caramel coloring 
(E150a), along with a glass of dried olive leaf tea and natural mineral water at a refrigerator 
temperature of ~4°C (intervention group B). In all groups, the meal (control or intervention) 
will be consumed first and the liquid (control or intervention) will be consumed immediately 
afterwards. The total time for consuming the meal (biscuits) will be 15 minutes and in the 
next 10 minutes, the liquid (olive leaf tea or water with caramel coloring) must also be 
consumed. After 7 days (washout period), the same volunteers will come to the specially 
designed area and the procedure will be repeated, with the difference that the control group 
of the 1st day will belong to intervention group A, intervention group A will belong to 
intervention group B and intervention group B will belong to the control group. Finally, on 
the 15th day, the procedure will be repeated with the same volunteers, where now the 
control group of the 1st day will belong to intervention group B, intervention group A will 
belong to the control group and intervention group B will belong to intervention group A. On 
all 3 days, blood samples will be taken from participants before, as well as 60, 120 and 240 
minutes after consuming the meals. The results of the analyses will be statistically processed 
to present data regarding postprandial levels of selected biomarkers and plasma antioxidant 
status. 
✓ Theoretical background and study objectives

2.1. Argument, Research evidence, Significance of study 
According to the scientific background to date, indices of postprandial lipemia, glycemia and 
oxidative stress appear to be associated with various chronic diseases, especially 
cardiovascular diseases (Koutelidakis and Dimou, 2016). A diet rich in highly processed, 
calorie-dense foods, which are also devoid of nutrients, often leads to excessive postprandial 
spikes in blood glucose and lipids. This condition, called postprandial dysmetabolism, causes 
immediate oxidative stress, which increases directly in proportion to the increases in glucose 
and triglycerides after a meal. The transient increase in free radicals triggers acute 
atherogenic 
changes, 
such 
as 
inflammation, 
endothelial 
dysfunction, 
blood 
hypercoagulability and sympathetic hyperactivity. Postprandial dysmetabolism is an 
independent predictor of future cardiovascular events even in non-diabetic individuals 
(O’Keefe et al., 2008). In response to this fact, studies have been conducted to investigate 
the postprandial effect of functional ingredients and foods on biochemical markers and 
markers of inflammation. 
Taking olive leaf extract, rich in antioxidants and polyphenols, may be able to attenuate the 
increase in glucose, triglycerides and inflammation after a meal, positively affecting 
postprandial dysmetabolism. Olive leaves, being rich in antioxidants, without adding 
additional calories, when added to a high glycemic index meal, may reduce the postprandial 
glucose passage into the blood, partly by slowing gastric emptying. Clinical studies that study 
the effect of olive leaf tea on human health are mostly long-term, as a result of which there 
is no scientific data available on the effect of its administration at a post-prandial level. 
Considering the above, this clinical study aims to investigate a possible post-prandial effect 
of products prepared with dried olive leaves, on selected biomarkers and on the antioxidant 
status of plasma, in healthy volunteers. 
Study Objectives / Research Questions 
Main Objective 
The aim of the study is to investigate the postprandial effect of products (biscuits and tea) 
with dried olive leaves, on indicators of postprandial lipemia, glycemia and oxidative stress in 
the blood (plasma antioxidant capacity, cholesterol, LDL, HDL, triglycerides, glucose, insulin, 
CRP, etc.). 
Research Questions 
The research questions of the present clinical study are the following: 
✓ What is the postprandial effect of consuming tea with dried olive leaves on the 
aforementioned blood biomarkers? 
✓  Does the postprandial effect of tea with olive leaves differ from the postprandial 
effect of the biscuit enriched with olive leaf powder, with regard to the biomarkers 
under examination? 
✓ Can the consumption of olive leaf tea or an olive leaf cookie, before a meal rich in fat 
and carbohydrates (cookies with flour, butter and sugar), statistically significantly 
affect postprandial glycemia, lipemia, inflammatory response? 
✓ Could the inclusion of olive leaf powder, in a cookie rich in carbohydrates and fats, be 
a practice to reduce spikes in glycemia, lipemia and oxidative stress? 

✓  Summary presentation / study design 
Summary 
A nutritional intervention - clinical study of cross-over design (cross over design: acute study) 
will take place in a sample of healthy volunteers. This specific research will be carried out 
in the specially designed area of the Human Nutrition Unit of the University of the 
Aegean in collaboration with an external physician who will undertake the work of blood 
sampling. 
Study design 
 In a total sample of approximately 35 healthy volunteers, the number of which has been 
determined through a special statistical program (G-power, see 4.1.) for calculating the 
required sample of the research to draw safe and reliable conclusions and in which a 
possible dropout of 10-15% has been taken into account, a nutritional intervention - clinical 
study of crossover design will be carried out. The study for each participant will last 15 days. 
Each participant on the first day in a 12-hour fasting state will be invited to visit between 
08:30 am and 13:00 pm, the specially designed blood collection room of the Human 
Nutrition Unit, Department of Food Science and Nutrition, housed in the building on 
Dimokratias Avenue of the University of the Aegean. In this specially designed space, 
questionnaires will be completed (24-hour recall), blood pressure will be measured for each 
volunteer (3 times in total on the right arm) and blood will be taken by catheterization 
(baseline) by a doctor. Afterwards, a meal will be served that will include 2-3 pieces of 
biscuits (with or without olive leaves) and 1 cold (~4°C) tea that will have been prepared with 
or without olive leaves. The consumption of each meal will take place within a period of 15 
minutes and in the next 10 minutes, the tea must also be completed by all participants. 
Finally, the following 
 
blood and blood pressure samples will be taken for the volunteers, which will be carried out 
60, 120 and 240 minutes (min) after the completion of the meal. 
After a seven-day washout period, on the eighth day, the same participants will be asked to 
return to the specially designed area of the University of the Aegean, at the previously 
determined hours (08:30am-13:00pm) to repeat the nutritional intervention and blood 
sampling process. Finally, on the fifteenth day, the same volunteers will return to the 
specially designed area of the University of the Aegean, at the previously determined hours 
(08:30am-13:00pm) to repeat the same process as on the first day. That is, a discussion will 
take place with the volunteer, questionnaires will be completed, their blood pressure will be 
measured and blood will be taken by catheterization (baseline) by a doctor. The meals that 
will be served to the volunteers are determined by the control or intervention group to 
which they belong. After each solid meal (biscuits) is consumed within 15 minutes and the 
liquid meal (tea) follows in the next ten minutes, the next blood pressure measurements and 
blood sampling will be performed for the participants 60, 120 and 240 minutes (min) after 
completing the meal. 
✓ Time point t0 = 0 minutes (min): Blood pressure measurement and taking a 10 ml 
blood sample in a fasting state and before taking any food, by venipuncture 
(Baseline). 

 
✓ Time point t1 = 25 minutes (min) / Meal consumption: 
Control group: 2-3 pieces of biscuits with caramel coloring (total 120 gr.) and a glass (250 mL) 
of tea (natural mineral water with caramel coloring) for the control group or

Intervention Group A: 2-3 pieces of biscuits with 1.25 g. dried olive leaf powder (total 120 g.) 
and a glass (250 mL) of tea (natural mineral water with caramel coloring) for intervention 
group A and  
Intervention Group B: 2-3 pieces of biscuits with caramel coloring (total 120 g.) and a glass 
(250 mL) of tea (natural mineral water with 1.25 g. dried olive leaves) for intervention group 
B. Solid meals (biscuits) should be consumed within 15 minutes (min) and tea intake should 
be completed within the next 10 minutes. 
✓ Time point t2 = 60 minutes (min), after the end of the meal: Blood pressure 
measurement and taking a 10 ml blood sample 60 minutes (min) after consuming 
the meal. 
✓ Time point t3 = 120 minutes (min), after the end of the meal: Blood pressure 
measurement and taking a 10 ml blood sample 120 minutes (min) after consuming 
the meal. 
✓ Time point t4 = 240 minutes (min), after the end of the meal: Blood pressure 
measurement and taking a 10 ml blood sample 240 minutes (min) after consuming the 
meal. 
Participant registration and case of study withdrawal 
The goal is to gather an initial sample of individuals (at least 40-50) from Greece in order 
to collect the required number of cases for the study. The research team will then 
proceed to examine the inclusion/exclusion criteria and select the participants to whom 
the meals will be provided, on the three different days of the study. 
Sample size calculations 
The sample size was calculated using G*power 3.1 (University of Düsseldorf, Germany). 
The sample size calculation revealed that 30 subjects were sufficient to detect a 
statistically significant group-time interaction, with an effect size of 0.25, at the 5% 
significance level with a 95% probability. Taking into account a dropout rate of 15%, the 
final sample size reached 35 subjects 
Inclusion criteria 
The criteria that those interested in conducting the study must meet are the following: 
▪ 
Healthy, without imminent diseases (e.g. diabetes, cardiovascular, hypertension, 
hypercholesterolemia, hypertriglyceridemia, morbid obesity with BMI> 40 kg/m2) 
▪ 
Aged 18-65 years at the start of the study 
▪ 
Signed written consent to participate in the study and access by doctors to their medical 
history (diseases, medication) 
 
Exclusion criteria 
Participants who meet the following exclusion criteria at the initial stage will be excluded 
from the study for reasons of safety, as well as for the effectiveness of the study: 
▪ 
 Individuals with a BMI >40 

▪ 
 Professional athletes 
▪ 
 Individuals taking contraceptives in the last 3 months 
▪ 
 Individuals with a history of food allergy with hypersensitivity to any of the components 
of the administered product (e.g. milk protein or allergy-hypersensitivity to plants of the 
Oleaceae family) 
▪ 
Individuals taking supplements (e.g. antioxidants) or medications that affect bowel 
function (e.g. antibiotics) in the last 3 months or medication (hypoglycemic drugs or 
antihypertensive drugs or diuretics) that may interact with the administered product 
▪ 
 Individuals taking any medication or supplement that may affect metabolism (e.g. GLP-1 
analogues and receptor agonists, caffeine supplement, weight loss/gain supplements) 
▪ 
 Pregnant, lactating or menopausal women 
▪ 
 Individuals with substance abuse or chronic alcoholism or total daily alcohol intake > 50 
g/d 
▪ 
 Individuals with a chronic condition (history of cardiovascular disease, cancer, diabetes, 
hypertension, hyperlipidemia, neurological and neuropsychological disorders, active liver 
disease, severe renal dysfunction, severe stroke and conditions associated with increased 
risk of bleeding) 
▪ 
 Individuals with any other serious medical condition that may affect the individual's 
ability to participate in a nutritional intervention study. 
▪ 
Individuals whose dietary preferences do not allow them to participate (e.g. vegetarians, 
vegans) 
▪ 
Individuals who are not willing to follow the study protocol and the fasting and eating 
periods 
▪ 
 Individuals who are considered unreliable by the researcher or have a shorter life 
expectancy than the expected duration of the study, due to an illness or if they are in any 
condition that in the opinion of the researcher does not allow their safe participation in 
the study (drug addiction, alcohol abuse) 
 
Strategy for the inclusion and retention of participants

▪ 
The study participants will be recruited by the University and the wider local community 
of Lemnos. The volunteers will be either students or members of the teaching staff of the 
University of the Aegean or residents of the island. One of the members of the research 
coordinators will undertake the briefing of each healthy volunteer, which will take place 
in a special area of the University. Those who meet all the study criteria to be accepted 
and at the same time agree in writing to participate in it, can participate in the study. On 
the days they come to this special area, they will have the opportunity at any time to ask 
for help either from the principal investigator or from any other representative of the 
research to record any difficulties or questions they may have. The members of the 
research team will be responsible for promptly informing the participants, in writing or by 
telephone, about the visits and the biological samples that need to be collected. 
▪ 
The scientific director of the study or his/her representatives will be responsible for the 
safekeeping of the data and results. The anonymity of the participants will be maintained 
and the safe storage of the history of the healthy subjects will be ensured, to which only 
the personnel who will be employed in this research will have access. 
 
Participant Withdrawal Criteria 
Participants are free to withdraw from the study at any time they wish. Accordingly, the 
investigator will be able to ask the participant to leave the study if: 
o Clinical adverse events, laboratory abnormalities, or other medical conditions or 
conditions are identified that would compromise the participant's continued participation 
in the study 
o Participants meet one of the aforementioned exclusion criteria (whether newly 
developed or previously unrecognized) that precludes further participation in the study 
o Participants appear to be inadequately adhering to the protocol requirements 
Suspension or early termination of the study 
This study may be suspended or terminated prematurely only if there is a sufficient reason. 
A written notice documenting the reason for the interruption or suspension of the study 
will be provided by the person responsible for this decision (research team, funding 
agency, hospital). 
 
Study Intervention 
✓ Intervention Description 
The meals will be given to the volunteers at the Nutrition and Public Health laboratory of the 
Human Nutrition Unit, University of the Aegean. 
For olive leaves, there does not seem to be any data suggesting that at these doses it may have 
a potential risk or negative impact on health, as on the one hand there is no confirmed 
adverse effect of its individual components on health based on the scientific literature and 
on the other hand, according to corresponding research on similar foods, their possible 
beneficial role in promoting health emerges. 
✓ Assessment of participant compliance for the administration 
The assessment of the compliance of the study participants with regard to the administration of 
meals and compliance with the required study criteria during blood sampling, of the 

volunteers who will come to the University premises to consume all the meals in the 
correct order and at the correct time, will be achieved after continuous supervision by a 
member of the research team. 
✓ Study Procedures 
✓ Questionnaires 
A specially designed questionnaire, based on bibliographic data, will be used to collect 
important data regarding the study participants. 
The questionnaire will concern the clinical status and habits (especially dietary) of the 
volunteers of the clinical study and will include the following parts: 
Part 1 
General descriptive data of the study population (gender, age, anthropometric indicators) 
Part 2 
Clinical status of each participant (medication, nutritional supplements, etc.) 
Part 3 
Dietary and other habits before the start of the intervention 
In addition, study participants will be given a 24-hour questionnaire, each day they attend for 
blood draws. 
✓ Biological samples 
The collection of blood samples, by a competent doctor, from healthy volunteers of the 
clinical study will be used to determine indicators of postprandial lipemia, glycemia and 
antioxidants in the blood (antioxidant capacity of the plasma, lipid profile and specifically 
triglycerides, HDL & LDL cholesterol, total cholesterol, insulin, glucose, hs-CRP, etc.) which will 
be analyzed using a special biochemical and immunological analyzer as well as in vitro 
laboratory methods. 
✓ Statistical analysis 
After the above analyses are carried out using a special biochemical and immunological 
analyzer, the use of the statistical package SPSS (Statistical package for social sciences: 
SPSS) will follow in order to record, process and analyze the results of the study. 
✓ Retention of study records 
The study records will remain registered for a maximum of 3 years from the date of their 
registration in special areas of the Human Nutrition Unit, University of the Aegean. Study 
records should be kept for a maximum of 2 years after the last marketing authorization or 
for longer if required by local regulations. 
✓ Ethical issues, Consent and Privacy Statement 
 Ethical standards 
The members of the research team agree to provide their professional expertise and 
knowledge for the conduct of the study in accordance with the requirements of the 
protocol and in accordance with the provisions of the Declaration of Helsinki and the 
provisions of the General Data Protection Regulation. 
 Research ethics and conduct rules 

During the conduct of this specific clinical study, all fundamental rules of ethical, scientific and 
research ethics will be observed. Therefore, an application for approval of the clinical study 
will be made by the Bioethics and Research Ethics Committee of the University of the 
Aegean, with the research volunteers also being informed of its purpose, the 
confidentiality of the data and the voluntary nature of their participation. Participants will 
take part in the study, after signing a declaration of consent to participate in it. 
Formal consent process 
Formal consent in the clinical study is a process that will begin before the registration of the 
interested parties in the study and will continue throughout their participation in it. 
Interested parties will be provided with a detailed discussion of the benefits and potential 
risks of the study, as well as a relevant consent form where the necessary procedures will 
be presented. Each interested party must read and review it or in special cases (e.g. blind) 
be informed by third parties. Interested parties will be provided with a copy. The 
investigator or representative will explain the study and its objectives to each participant 
and will resolve any questions they may have. 
The rights and care of interested parties will be protected by the fact that the quality of 
their clinical care will not be affected in any way if they refuse to participate in this clinical 
trial.