Short Term Turkish Coffee Consumption Elevates Cardiovascular Risk Markers, Decreases Leptin Hormone Levels, and Impairs Sleep Quality in Healthy Young Women: A Pilot Randomized Controlled Trial

Study Design and Participants This study was a pilot randomized, controlled, parallel-group clinical trial to examine the physiological impact of TC consumption over a four-week intervention period among healthy young women. The study was approved by the Institutional Review Board (IRB) at the University of Petra (UOP) with the ID E/H/5/10/2024.

The present study was retrospectively registered at the Open Science Framework (OSF, DOI 10.17605/OSF. IO/2V96N) because OSF is a free and open-access platform that allows transparency and academic cooperation and helps to share the research evaluation process https://osf.io/2v96n/?view_only=19a4815ef45f4ae0814c5a381071163b. This pilot study adhered to the ethical principles of the Declaration of Helsinki following the CONSORT 2010 guidelines for reporting randomized clinical trials, ensuring methodological rigor and transparency, a complementary checklist of CONSORT 2010 is provided supplementary file. After the aims and procedures of the study and the possible risks and rights of the research participants were explained, written informed consent was obtained from all participants. The study duration consisted of conducting participant recruitment and four-week interventions within a two-month period from August 2024 to October 2024.

A total of 50 healthy young females were initially recruited through digital advertisements, social media platforms, and printed flyers. The screening procedures involved a 20-minute face-to-face structured interview to assess eligibility and collect demographic and health-related data. The inclusion criteria were as follows: age (18-25 years), normal body mass index (BMI) (18.5-24.9 kg/m²), nonsmoking status, and infrequent caffeine intake (defined as ≤1 cup/week). Participants with chronic illnesses (e.g., diabetes, thyroid disorders, cardiovascular, or renal conditions), current medication or supplement use affecting appetite, metabolism, or sleep, as well as those following therapeutic diets or enrolled in other clinical trials, were excluded because they met the criteria for prior coffee intervention trials. Those who self-reported as healthy and who met the inclusion were chosen. To control for residual caffeine effects and standardize baseline conditions, a three-week caffeine washout phase was implemented prior to the intervention. During this period, participants were instructed to avoid all caffeine-containing products (coffee, tea, chocolate, soft drinks, energy drinks, and supplements). Adherence was verified through daily self-report logs and unannounced follow-up calls. It is the same strategy, as the previous coffee intervention study protocols washout adherence was used on the basis of subject instruction and self-observation. Three participants (n=3) who consumed caffeine during this phase were excluded from the study.

Following successful washout, 47 participants remained eligible and were randomized via stratified block randomization (based on BMI) into either the intervention group (TC) or the control group. However, prior to initiating the intervention, seven participants (n=7) withdrew voluntarily for personal reasons or scheduling conflicts. The final sample that commenced the intervention comprised 40 participants: 20 assigned to the TC group and 20 assigned to the control group, as shown in Figure 1. The randomization sequence was generated via Randomization.com and administered by an independent researcher to maintain allocation concealment and reduce selection bias.

2.2. Intervention protocol and coffee samples After the washout period and randomization, participants in the intervention group were given a directive to drink TC daily for a period of four weeks, whereas participants in the control group discontinued intake of TC (abstinence) and continued with their usual diet and lifestyle. Owing to the nature of the intervention, blinding was feasible, as it is not possible to find an alternative drink similar in its sensory attributes such as color, smell and flavor that mimics the attributes of the traditional Turkish coffee to use it as a placebo. However, all participants followed the same standardized instructions not to drastically modify dietary patterns during the study. In this study, a sufficient quantity of finely ground Brazilian Arabica TC, consisting of an equal blend of medium and dark roasted coffee beans (5 grams each), was purchased. This is the type of coffee commonly consumed by Jordanian consumers, as reported in studies on Jordanian coffee preparation and drinking habits. The desired amount was distributed to each participant for a period sufficient for the study period, along with optimal storage, preparation, and consumption instructions. The traditional Jordanian way of preparing Turkish coffee was followed by mixing 5 grams of finely ground coffee steeped in 55 mL of cold water in a Rakwa (traditional Turkish coffee pot). The foam on the surface was formed by heating the mixture slowly over low heat. The same amount of boiled plum was approximately 40 mL/serving, as per the national standard consumption size. The caffeine content and bioactive compound exposure per cup were calculated using this standardized serving volume. To approximate daily consumption habits and maintain the integrity of coffee components, this unfiltered preparation method was used while preserving bioactive compounds, such as chlorogenic acids and diterpenes. Each participant consumed three daily cups (40 mL each) of TC throughout the day over three specified time periods: the first cup between 7 and 9 AM, the second cup between 12 and 2 PM, and the third cup between 3 and 4 PM, with evening consumption strictly prohibited. The study adopted this protocol through morning, midday and early-afternoon coffee consumption because research has shown that caffeine consumed 6 h before bedtime disrupts sleep quality. Infinitely tracked protocol compliance through daily logs and calls to its participants. The beverage was consumed with no additives (except where the subjects habitually used sugar, these subjects continued their habitual sugar quantity to avoid changing their habitual consumption). Coffee packets were preportioned, and each participant was given preparation instructions along with calibrated utensils to ensure standardization and compliance. In the laboratory, the caffeine concentration in the prepared coffee solution was analyzed and determined to be approximately 1.77 mg/mL via high-performance liquid chromatography (HPLC) coupled with a diode array detector (DAD) and mass spectrometer (MS) and was found to be equivalent to approximately 71 mg of caffeine/40 mL cup. This approach was consistent across all consumed servings and permitted precise quantification of the bioactive load. Daily intake logs and random follow-up phone calls were used to monitor participant compliance. Furthermore, at baseline and postintervention, 24-hour dietary recall was standardized through statistical analysis for potential confounding variables such as physical activity and dietary intake.