Comparing the Effect of High-Fat (Peanuts) Versus High-Protein (Plain Greek Yogurt) Snacks on Appetite Hormones and Satiety Among Overweight and Obese Women
Phase: NA
Conditions: Body Weight, Appetitive Behavior, Dietary Exposure, Hormone Deficiency
Sponsor: Al-Balqa Applied University

HIGH FAT VS HIGH PROTEIN AND APPETITE HORMONES 
 
 
NCT # 04518930 
Document Date: 03/06/2024 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Study protocol  
 
Study design and participants 
 
The study design utilized a parallel-arm intervention, employing a non-blind randomized clinical 
trial format with two distinct treatment groups. This study enrolled fifty-two healthy Jordanian 
women from a private nutrition clinic in Amman through randomization. 
This study was conducted in accordance with the ethical guidelines outlined in the Declaration of 
Helsinki. The research procedures involving study participants received approval by the 
Institutional Research Board committee at the University of Jordan (No. 19/2021/180). 
Furthermore, the study was registered on ClinicalTrials.gov (No. NCT 04518930). Written 
informed consent was obtained from all individuals who participated in the study. 
 Inclusion and exclusion criteria  
The study's inclusion criteria encompassed women aged between 30 and 40 years, with a 
body mass index (BMI) ranging from 25 to 35 kg/m². The included age group comprises 
predominantly overweight and obese individuals attending the nutrition clinic, 
demonstrating high adherence to the study protocol. These individuals engaged in light 
exercise 1-3 times per week (activity factor 1.375) and adhered to a routine of consuming 
three regular meals, along with two daily snacks. They maintained a water intake of eight 
cups and ensured a sleep duration of  8 hours. Additionally, participants were required to 
have no known allergies, particularly to nuts, especially peanuts and yogurt. 
Conversely, women were ineligible for the study if they met any of the following 
conditions: age below 30 years or above 40 years, BMI below 25 kg/m² or above 35 kg/m², 
engaged in little or no exercise or engaged in moderate to heavy exercise , were pregnant 
or lactating mothers, had allergies to nuts, peanuts, yogurt, milk, or milk products, were 

using medications, hormonal therapy, supplements, oral contraceptives, or herbal/botanical 
products claimed to suppress appetite. Furthermore, exclusion criteria encompassed 
women with hormonal disturbances, including those in a menopausal state, and those 
within their menstrual cycle days or within one week prior, commonly experiencing 
hyperphagia due to elevated progesterone levels. Additionally, individuals with chronic 
diseases and metabolic disorders such as cardiovascular diseases, diabetes, hypertension, 
kidney diseases, thyroid disorders, gastrointestinal diseases, polycystic ovary syndrome, 
or androgen disorders were excluded, following a weight-reducing diet, experiencing sleep 
disorders, or sleeping less than 8 hours per day, and consuming less than 8 cups of water 
per day were also excluded from the study. 
 
Sample size determination 
The required sample size for the study was determined using the formula:  
n = 2 * (Zα + Zβ)2 * SD2 ÷ δ2, where n represents the required number of participants in both the 
treatment and placebo groups. Zα and Zβ are the values from the standard normal distribution 
corresponding to specific confidence levels of 95% and a two-sided α of 0.05 (Zα = 1.96) and a 
power of 80% (Zβ = 0.84). SD refers to the standard deviation (pooled), while δ represents the 
estimated difference between the treatment and placebo groups. 
Based on the above equation, the sample size calculation indicated that approximately 20 subjects 
were needed for each arm of the trial to detect a change of 1.76 μU/mL in insulin levels between 
the treatment and placebo groups, with 80% power and 5% significance. The standard deviation 

(SD) was assumed to be 2.1. Therefore, n = 2 * (1.96 + 0.84)2 * (2.1)2 ÷ (1.76)2 ≈ 22 participants 
per group. 
To account for potential participant dropouts and to enhance the statistical power of the analysis, 
the number of participants was increased to 26 women per group. 
Overview of the study design and  procedures 
The study employed a two-arm parallel design, consisting of 52 participants who were randomized 
into two groups. The Greek yogurt group (n = 26) received one serving (380 g, 200 kcal) of zero-
fat Greek yogurt, and the peanut group (n = 26) received one serving (35 g, 200 kcal) of unsalted 
peanuts. Due to one participant from each group dropping out and not attending the second visit, 
only 25 subjects were included in the study analysis in each group. 
All participants attended two visits to the clinic. During the initial visit, participants were instructed 
to arrive fasting for 10-12 hours, during which anthropometry and body composition analysis were 
conducted. They also received education and instructions to follow a three-day low-calorie diet 
totaling 1200 kcal, comprising breakfast, snack 1, lunch, snack 2, and dinner, prior to the 
interventions. On the second visit (post-diet visit), participants received and consumed their 
snacks, completed the visual analog scale (VAS) questionnaire and blood samples were 
withdrawn. 
Treatment/intervention and randomization 
Intervention 
Two distinct interventional snacks were provided to the participants: plain Greek yogurt and 
peanuts. In the Plain Greek yogurt group (n=25), each participant consumed 380g of plain Greek 

yogurt, equating to approximately 200 calories. In the peanut group, (n=25) each participant 
consumed 35g of roasted, unsalted peanuts, providing approximately 200 calories. 
The plain Greek yogurt (non-flavored /30g protein/13.5g carbohydrate/0g fat) was procured from 
the supermarket and stored in the refrigerator until serving. The peanuts (roasted, unsalted, with 
skin/ 8.4g protein/7.6g carbohydrate/17.5g fat) were purchased one day prior to the serving date 
to preserve their attributes. They were meticulously prepared in a safe and sterile manner, 
portioned into small transparent pouches, each containing 35g. These packed peanuts were then 
stored in a dry, room-temperature environment until the serving date. Table 1 shows the macro 
and micronutrients composition of the two interventional snacks. 
The intervention was provided to each participant upon arrival at the clinic based on pre 
randomization and they were asked to consume it at 1:00 pm.  
Randomization 
The participants were assigned to two groups in sequentially, following a predetermined 
randomization list. “Peanut”  and “Greek yogurt” were each written clearly in Arabic on  26 folded 
papers. The list was created by randomly selecting folded papers from a basket, each containing 
either the label "peanut," or "plain Greek yogurt". The groups were then  identified as the peanut 
group and the Greek yogurt Group based on the random selection of each participant . 
All participants adhered to a 1200 kcal diet plan, including  breakfast, snack 1, lunch, snack 2, and 
dinner. This diet plan remained consistent for all participants over three consecutive days, except 
during the second visit, where specific snacks were provided based on the randomization list.  
Data collection and assessment 
Pre-diet visit 

During the initial visit, participants were provided with a three-day low-calorie diet menu totaling 
1200 kcal. This menu included breakfast, snack 1, lunch, snack 2, and dinner, along with a follow-
up sheet. They were instructed to adhere to the foods listed in the diet menu and to refrain from 
consuming peanuts or any peanut-based products, as well as Greek yogurt, during snack 1 and 
snack 2. Additionally, they were briefed on the necessity of daily follow-up via phone to monitor 
their compliance with the given instructions. Body composition analysis was performed using 
bioelectrical impedance analysis (Inbody 770). To categorize participants into groups, they were 
randomly allocated based on a pre-determined randomization list. 
Anthropometric and body composition analysis 
Before the body composition analysis test, each participant visited the clinic after overnight 
fasting for a period of 10-12 hours. The analysis was conducted using bioelectrical impedance 
technology, which automatically recorded several parameters, including weight, height, BMI, 
skeletal muscle mass, soft lean mass, fat mass, and abdominal circumferences. To ensure precise 
readings, participants were advised to wear lightweight clothing and remove any metal items, 
such as accessories, belts, money, and socks before the test. 
Post-diet visit 
On the second visit, scheduled after three days of adhering to the 1200 Kcal diet, all participants 
arrived at the clinic at 1:00 pm. Prior to their arrival, they had breakfast at home at 9:00 am. Upon 
arrival, a trained healthcare professional collected baseline blood samples from each participant. 
At 1:00 pm, participants in the plain Greek yogurt and peanut groups consumed their assigned 
snacks. Following snack consumption, a validated VAS questionnaire was distributed to assess 
sensations and appetite levels at 0 minutes (1:00 pm), 30 minutes (1:30 pm), and 60 minutes (2:00 

pm). Additionally, one hour after their arrival, at 2:00 pm, a second blood sample was collected 
from all participants. Throughout this period, participants were situated in a stress-free 
environment designed for relaxation and comfort, with soft music played to enhance their 
experience. No adverse events were reported by any of the participants following snack 
consumption. 
Appetite perceptions 
Appetite sensation was evaluated using a validated VAS questionnaire, designed to measure 
subjective ratings of hunger, fullness, satiety, prospective food consumption, and desire to eat. The  
VAS questionnaire included questions assessing “how strong is your feeling of” hunger, fullness, 
and desire to eat or “how much food can you eat right now” with anchors of “not (much) at all” to 
“extremely/an extreme amount”. 
 Participants were instructed to indicate their sensations on the scale at various time points during 
the study: upon arrival, 30 minutes, and 60 minutes after consuming the snack. This allowed us to 
discern the impact of consuming Greek yogurt and peanuts on appetite perceptions over time. The 
decision to assess at 30 and 60 minutes was based on the rapid increase in gut hormones following 
food intake, with their secretion peaking around 1-2 hours later. The VAS questionnaire was self-
administered, ensuring a convenient and straightforward assessment for the participants. 
 
Biochemical analysis 
The collected blood samples ( baseline and after 60 minutes of intervention) were labeled with a 
unique code to ensure proper identification before being transported to the medical laboratory.  

The blood samples were drawn into plain tubes containing separation gel and left undisturbed until 
coagulation occurred. After coagulation, serum was separated from the blood cells through 
centrifugation at 882 xg for 20 minutes. The resulting serum was then carefully transferred into 
Eppendorf tubes and stored at -20 °C to preserve the integrity of the peptides for subsequent 
analysis. These storage procedures adhered to the recommendations outlined in studies conducted 
by Hallworth and Lobely. 
All biochemical blood assays were conducted in duplicate, and the average of these duplicate 
measurements was utilized in the statistical analysis. 
CKK, PYY, GLP-1 and insulin analysis 
The blood samples were sent to the laboratories of Aurum Biotechnology Company, where the 
researcher conducted the analysis following the instructions provided in the respective manuals. 
The analysis utilized the following ELISA  kits: Human PYY ELISA  kit (Lot No: 9680012101) 
(catalog no. RK02174; ABclonal, USA), Human GLP-1 ELISA  kit (Lot No: 9680012107) 
(catalog no. RK09098; ABclonal, USA), Human CCK ELISA  kit (Lot No: 1210528141) (catalog 
no. MBS770851; MyBioSourceA) and Human Insulin ELISA  kit (lot No: 9680004240821) 
(Catalog. no. RK 00302; ABclonal, USA). 
Ghrelin analysis 
The analysis of GHRL (ghrelin) was carried out using the Human GHRL ELISA  kit (Lot No: 
38401422) (catalogue no. MBS2602099; MYBioSource, USA). This kit utilizes the Competitive-
ELISA  method. The well plates used for the analysis were pre-coated with GHRL. In the 
reaction, the GHRL present in the sample or standard competed with a fixed amount of GHRL 
on the solid phase supporter for binding sites on the Biotinylated Detection Antibody specific to 

GHRL. 
 
Statistical analysis  
The statistical analysis was conducted using SPSS version 25.0 (IBM SPSS Statistics for 
Windows, IBM Corporation). Before analysis, a nonparametric Kolmogorov-Smirnov test was 
employed to assess the normal distribution of all continuous variables. Descriptive statistics were 
used to assess the frequencies of categorical variables, while means ± standard deviations (SD) 
were calculated for continuous variables.  
To determine differences between the means of normally distributed continuous variables in both 
the plain Greek yogurt and peanut groups, a Student t-test for independent samples was 
employed. Additionally, a paired sample t-test was used to assess variations in means of normally 
distributed variables before and after snack consumption in both groups. In instances where 
variables displayed significantly skewed distributions, medians were compared using the Mann-
Whitney U test for independent samples, while paired samples were compared using the 
Wilcoxon Signed Rank test. A two-way repeated measures ANOVA was performed to test the 
main and interaction effects of Greek yogurt, peanuts, and time on appetite and insulin hormones. 
Additionally, the repeated measures procedure of the General Linear Model (GLM) was utilized 
to analyze the impact of plain Greek yogurt and peanuts on appetite perceptions over time. 
Correlations between gut hormones, insulin, and anthropometric measurements were assessed 
using the Pearson two-tailed bivariate test for continuous variables. All p-values are two-tailed, 
and a significance level of p < 0.05 was considered.