Exercise Effects on Appetite-regulating Hormones and Cardiovascular Risk Factors in South Asian and White European Men
Conditions: Risk Factor, Cardiovascular, Physical Activity, Pre-diabetes
Sponsor: Kingston University

Title Page 
Project Title: Exercise Effects on Appetite-regulating Hormones and Cardiovascular Risk 
Factors 
NCT number: TBC 
Date of Document: 02/10/18 
 

Preliminary measures 
Prior to the main trials, participants will visit the human physiology laboratory to undergo 
screening, familiarisation, anthropometric measurements and a maximal cycling exercise test 
to determine maximum oxygen uptake (V̇ O2 max). Body composition will be assessed using 
air displacement plethysmography (BodPod; software version 5.2.0, COSMED, Rome, Italy) 
and waist circumference will be measured in duplicate to the nearest 0.1 cm at the midpoint 
between the xiphoid process and the iliac crest using a standard anthropometric measuring tape 
(HaB International Ltd., Southam, UK). Blood pressure will be measured using a digital 
monitor (Omron M10-IT, Omron Healthcare Co. Ltd., Japan). After familiarisation with the 
testing equipment, participants will perform an incremental exercise test to volitional 
exhaustion on an electromagnetically braked cycle ergometer (Lode Excalibur Sport, 
Groningen, Netherlands) for the determination of maximum oxygen consumption (V̇ O2 max). 
Participants will cycle at a self-selected pedal rate between 70 to 90 revolutions per minute for 
3 min at 80 watts (warm up), followed by increments of 30 watts every 3 minutes until 
volitional fatigue. Expired air samples will be monitored continuously using an online breath-
by-breath gas analysis system (Oxycon Pro, Viasys Healthcare Gmbh, Höchberg, Germany). 
Heart rate (Polar T31; Polar Electro, Kempele, Finland) and ratings of perceived exertion 
(RPE) will be also monitored throughout the test. The calculated V̇ O2 max will be used to 
determine the relative intensity of exercise during the main trials.  
Main trials 
Participants will complete two, 2-day trials (exercise and control) in a random order separated 
by an interval of at least 1 week. Each trial will require participants to attend the laboratory for 
7 hours. On day 1 of the trials, participants will arrive at the human physiology laboratory 
between 8.00 am and 9.00 am. Upon arrival at the laboratory, a fasting venous blood sample 

will be collected for the measurement of appetite-related hormones, inflammatory markers and 
metabolic profiling. The 7 h trial will commence upon collection of the first blood draw (0 h).  
After collection of the fasting blood sample, participants will consume a standardised breakfast 
containing bread, tuna or ham, muffin and orange juice. Another blood sample will be collected 
30 minutes after breakfast. Sixty minutes of continuous cycling at 65 % maximum oxygen 
uptake will be performed using a bicycle ergometer 2 hours after consuming breakfast. Before 
and after performing the exercise, a blood sample will be collected. Heart rate and RPE will be 
also monitored during the exercise. One hour after completing the exercise, participants will 
be given 30 minutes ad libitum access to a buffet meal containing white bread, margarine, 
butter, mayonnaise, ham, tuna, cheese, chocolate bars, whole fat milk, corn flakes, muesli, 
granola, orange juice, chocolate muffin, apple, banana, oranges and water. Before and after this 
meal, a blood sample will be collected. After finishing the meal, participants will rest until the 
end of the trial (sitting reading, working or watching movies). Another blood sample will be 
collected before participants will leave the laboratory (7 h). Appetite perceptions will be also 
assessed at baseline and every 30 min during the trial using validated 100 mm visual analogue 
scales (VAS). The procedures in the control trial will be identical to the exercise trial except 
that no exercise will be performed.  Blood samples and appetite perceptions will be collected 
at the same times as in visit 2. 
Blood sampling and biochemical analysis 
Blood samples will be used to determine metabolic markers, inflammatory markers and 
appetite hormones, including cell count, glucose, insulin, total cholesterol, HDL-C, LDL-C, 
TAG, CRP, TNF-α, IL-6, acylated ghrelin, PYY, Leptin and fatty acids. Commercially 
available enzyme-linked immunosorbent assays (ELISA), colorimetric methods and gas 
chromatography mass spectrometry (GS-MS) will be used for blood analysis.  

Blood samples will be collected into two pre-cooled 4 ml EDTA tubes, one 6 ml EDTA tube 
and one 6 ml heparin tube on 7 different occasions during the main trials for a total of ~ 140 
ml. Blood sampling will be performed by venepuncture and using a cannula inserted into an 
antecubital vein. These samples will be analysed and immediately disposed of in clinical waste 
or spun down to serum or plasma and stored at -80°C according to the Human Tissue Act 
(HTA). All procedures will be carried out by fully trained individuals who are experienced in 
conducting such protocols. The technical support will always be available and first aiders will 
be available on site when testing is taking place. All blood sampling will be performed by a 
trained phlebotomist. 
Statistical analysis 
All statistical analyses will be conducted using the analytical software SPSS version 23.0 for 
Windows (SPSS 23.0, IBM Corp, Armonk, NY, USA). Normality of the data will be checked 
using Shapiro-Wilk tests and not normally distributed variables will be natural log transformed 
before analysis. Absolute standardised effect sizes (ES) (Cohen’s d) will be calculated for each 
variable and statistical significance will be accepted as P < 0.05.