The study has been approved by the Medical Ethics Committee of Maastricht University Medical Center + (MUMC +), Maastricht, The Netherlands, and was conducted in full accordance with the Declaration of Helsinki (as amended by the World Medic Association in 2013) and the Dutch Regulations on Medical Research Involving Human Subjects (WMO 1998). All subjects gave their written informed consent before participation. This study has been registered at ClinicalTrials.gov (NCT02990637).
Healthy subjects, aged 18–70 years with a body mass index (BMI) between 25 and 35 kg/m2 and elevated cholesterol levels, were recruited by means of advertisements in local newspapers and posters on notice boards at the Maastricht University buildings and from an existing cohort of eligible subjects. For this study, we sought participants with baseline total cholesterol levels in the range of 5.0–8.0 mmol/L, based on the guidelines of the Dutch heart association (https://www.hartstichting.nl/). Cholesterol values within this range are classified as elevated, while values above 8.0 mmol/L are classified as markedly elevated and generally result in the prescription of cholesterol-lowering mediation. Exclusion criteria were: (history of) chronic or severe diseases that may affect study outcomes or limit participation in the study; use of medication influencing endpoints of the study; administration of investigational drugs or participation in any scientific intervention study which could interfere with the study; use of antibiotics within 30 days prior to the start of the study; use of antioxidants, minerals and vitamin supplements; pregnancy or lactation; abuse of alcohol (> 20 alcoholic units/week) or recreational drugs; smoking; recent weight gain or loss (> 3 kg in previous 3 months); high physical activity (> 4.5 h of running/week); history of any side effects towards intake of olives. In total, 109 subjects were assessed for eligibility. Of these, 77 subjects were included in the current study (Supplemental Fig. 1).
The study was designed as a randomized, parallel, double-blind, placebo-controlled trial, conducted at the Metabolic Research Unit of Maastricht University, Maastricht, The Netherlands. The study participants were randomly assigned to receive one of the following two interventions: placebo or OLE. The randomization list was computer-generated by an independent person, with random and concealed block sizes of 2, 4, and 8 for treatment allocation. Participants and researchers were blinded to the intervention allocations until all analyses were completed. The total treatment duration was 8 weeks and measurements were performed at three different time points, i.e., at baseline (test day 1), after 4 weeks (test day 2), and after 8 weeks (test day 3) of treatment. A total intervention period of 8 weeks was chosen as this is in line with previous human intervention studies showing a significant improvement in blood lipid profiles after OLE supplementation and it has been suggested to be of sufficient duration to show a sustained effect on blood lipid levels as mentioned by the European Food Safety Authority [24, 25, 28]. The test day at 4 weeks was added to also assess the short-term effects of OLE supplementation on blood lipids. Before each test day, subjects were instructed to refrain from eating and drinking (except for water) after 10 pm in the evening. In addition, subjects were asked not to consume any alcohol-containing beverages and abstain from vigorous physical exercise from 2 days prior to testing. To limit the influence of diet, participants were asked to eat the same type of meal before each test day. Furthermore, subjects were instructed not to consume any foods containing olive phenolics during the entire duration of the study and to maintain their habitual diet. All measurements were performed in the morning in a quiet, temperature-controlled room (20–24 °C). After an overnight fast, subjects arrived at the study site, where they handed in a 3-day dietary record. Next, anthropometric (height, weight, and waist and hip circumference) and blood pressure measurements were performed. Then, blood samples were collected from an antecubital vein in the forearm and, finally, a questionnaire was completed to assess gastrointestinal symptoms, stool consistency, and stool frequency. Supplementation with the study product started after completion of the baseline measurements.
The primary objective of this study was to assess the effect of daily OLE supplementation on blood lipid profiles as measured by serum levels of total cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, and triglycerides. The secondary objective was to assess the effect of OLE supplementation on risk markers related to the development of CVD, which were lipid peroxidation as measured by plasma-oxidized LDL (oxLDL) and blood pressure. As explorative objectives, effects on glucose metabolism and liver function parameters were assessed.
The test product that was used was an extract prepared from olive leaves (Olea europaea L.) using a 100% water-based extraction method, standardized for its oleuropein content (> 16%), supplied by Interquim SA (Murcia, Spain). The batch used in the current study had an oleuropein content of 16.7%, providing 83.5 mg oleuropein per day. This dose was chosen based on previous studies showing a positive effect on blood lipid profiles after intake of OLEs [24,25,26]. As placebo, maltodextrin (Gonmisol, Barcelona, Spain) was used. The study product was provided as capsules, containing 350 mg of maltodextrin for the placebo or 250 mg of OLE in combination with 100 mg of maltodextrin. The extra 100 mg of maltodextrin was added to meet the minimum volume requirement of the capsules. Participants were asked to ingest two capsules each morning with 200 mL water 30 min before breakfast. The participants were instructed to return empty packages and unused study product during the last visit for compliance assessment.
Blood lipids, glucose, insulin, and liver function parameters
Serum levels of total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, and plasma levels of glucose were measured by spectrophotometry (Cobas 8000 analyzer series, Roche Diagnostics, Mannheim, Germany). Serum levels of insulin were determined using an immunometric assay (XPi instrument, Siemens Medical Solutions Diagnostics, LA, USA). Blood lipids, glucose, and insulin levels were determined at baseline, after 4 weeks, and after 8 weeks of supplementation. Liver function parameters were determined at baseline and after 8 weeks.
For analysis of oxLDL, 8 mL blood was collected in an EDTA tube at baseline, after 4 weeks, and after 8 weeks of supplementation. Samples were centrifuged within 30 min at 4 °C at 1250g for 10 min to obtain plasma, which was stored at − 80 °C until further analysis. OxLDL measurements were performed by a sandwich ELISA procedure according to the manufacturer’s instructions (oxLDL, Mercodia AB, Uppsala, Sweden).
Blood pressure and heart rate
Blood pressure and heart rate were assessed using a semi-continuous blood pressure monitoring device (Omron, Hoofddorp, The Netherlands) at the upper left arm after a 30-min rest in supine position. Four measurements were conducted at 5-min intervals. The first measurement was not used; the three other ones were averaged.
To assess dietary intake, participants were asked to complete a 3-day dietary record before each test day. Volunteers were asked to record the intake of 2 week days and 1 weekend day. Before the start of the study, participants were instructed on how to record their food and beverage intake, i.e., based on standard household units. During each test day, the records were checked, discussed with the participant, and completed in case of missing data. Energy and nutrient intake were analyzed using the online dietary assessment tool of The Netherlands Nutrition Centre (www.voedingcentrum.nl), which is based on the Dutch Food Composition Dataset (NEVO, National Institute for Public Health and Environment, Ministry of Health, Welfare and Sport, the Hague, The Netherlands).
Gastrointestinal tolerance, stool consistency, and stool frequency
Gastrointestinal tolerance was assessed using the gastrointestinal symptom rating scale (GSRS) as described previously . To assess defecation frequency and stool consistency, the Bristol Stool Form Chart was used .
The sample size calculation was determined for the primary outcome of the study, which was the effect of 8 weeks of OLE supplementation on blood lipid profiles. Based on a previous study in participants with normal cholesterol levels  and the assumption that a 5% stronger effect would be observed in the current study population, a sample size of at least 36 participants per group was required. A significance level of alpha = 0.05, a power of 80%, a difference in LDL-cholesterol of 0.4 mmol/L, and a standard deviation of 0.6 mmol/L were assumed for this calculation.
Statistical analyses were performed using IBM SPSS Statistics for Windows (version 25; IBM Corporation, Armonk, NY, USA). Baseline characteristics are presented as mean ± standard deviation (SD) for numerical variables and numbers for categorical variables. Analyses were performed on an intention to treat basis, including all subjects with baseline cholesterol levels between 5.0 and 8.0 mmol/L. Differences in blood lipid profiles, lipid peroxidation, blood pressure, heart rate, glucose metabolism, liver function parameters, anthropometrics, dietary intake, intestinal symptoms, stool consistency, and stool frequency were assessed by linear mixed model analyses (marginal model) with intervention group (OLE or placebo), time (baseline, 4 and 8 weeks), and intervention*time as fixed factors. For this model, an unstructured covariance structure for repeated measures was used. Results obtained with this model are presented as estimated mean ± standard error of the mean (SEM), with p values for the differences in means between groups after 4 and 8 weeks adjusted for baseline differences, where a two-sided p value ≤ 0.05 was considered statistically significant. For all outcomes, correction for multiple testing was performed by the false-discovery rate (FDR) of Benjamini–Hochberg based on a correction for multiple time points and parameters.