Extreme Exercise and Energy Expenditure (4E) Study
Phase: NA
Conditions: Cardiovascular Diseases, Obesity, Visceral, Liver Fat, Metabolic Syndrome, Lifestyle
Sponsor: Institut universitaire de cardiologie et de pneumologie de Québec, University Laval

 
 
Study Protocol including Statistical Analysis Plan 
 
 
 
 
Official Study Title: Extreme Exercise and Energy Expenditure 
(4E) Study 
 
 
 
NCT Number: N/A 
 
 
 
Document Date: June 13, 2018

BACKGROUND 
Although there is robust evidence that a physically active lifestyle including regular leisure 
time physical activity and exercise (>150 min per week of moderate to vigorous exercise 
per week) is associated with a reduced mortality risk and improved cardiometabolic health 
(1), some scientists have promoted the notion (which has certainly been taken up the lay 
press) that if one wants to lose weight, focus should be placed on caloric restriction rather 
than on exercise (2, 3). Several academic debates have been held on this issue (4, 5) 
which have left some health professionals and the public confused and perplexed (6). 
Our research group has been active in this area on several fronts. First, we have pioneered 
the notion that in cardiovascular and “metabolic” medicine, the health risk of overweight 
and obesity was not appropriately assessed by an index based on body weight (body 
mass index, which is expressed in kg/m2) (7-9). For instance, using imaging technology 
(computed tomography), we were among the first, more than 30 years ago, to begin to 
extensively study regional body fat distribution, showing that excess visceral adipose 
tissue accumulation was more closely related to an atherogenic and diabetogenic 
cardiometabolic risk profile than excess body weight or total body fat (7). As sedentary 
individuals with a diet of poor nutritional value (rich in processed fat and added sugar - 
promoting preferential visceral fat deposition) are also often characterized by a lower 
muscle mass and by high levels of abdominal visceral fat (9), several lifestyle intervention 
studies that we and others (10, 11) have conducted in viscerally obese sedentary men 
have shown that with regular exercise, one could lose visceral adipose tissue while gaining 
muscle mass, which would lead to trivial or sometimes no changes in body weight. Thus, 
if body composition is altered by an energy deficit produced by regular exercise, we have 
proposed that weight loss may sometime be misleading as an outcome as it may not 
always reflect favorable changes in body composition (muscle mass increasing) and in 
visceral adiposity and levels of ectopic fat (decreasing) (12). On that basis, we have 
proposed that changes in waist circumference could be more useful than body weight loss 
in the evaluation of the benefits of a lifestyle modification program involving regular 
endurance exercise such as brisk walking, jogging, or biking (12). For instance, we have 
previously reported that waist circumference was rather sensitive to lifestyle changes 
involving regular physical activity/exercise (10). Accordingly, lifestyle intervention studies 
have shown that a reduction of 4 cm in waist girth was predictive of a 58% reduction in 
the risk of developing type 2 diabetes among individuals who were initially characterized 
by abdominal obesity and glucose intolerance (13, 14). 
Thus, there is a need to propose a paradigm shift among health professionals to evaluate 
the impact of lifestyle intervention programs involving physical activity/exercise in high-
risk overweight obese individuals with visceral obesity: Weight loss is no longer the optimal 
or most clinically relevant outcome – losses of visceral adiposity and ectopic fat may be 
more relevant clinically. 
In 2015, a member of our team was asked to write an editorial (15) on a spectacular study 
which provided very relevant data to the debate about diet vs. exercise and weight loss. 
In that impressive exercise study, recreational middle-aged cyclists were required to bike 
2,700 km over 14 days and asked to try to match their energy intake to their very large 
energy expenditure, the objective being not to lose weight over the course of the 
intervention. Thus, the daily exercise “prescription” was huge as the authors wanted to 

3 
 
test the ability of these elderly and fit recreational cyclists to match their energy intake to 
their energy expenditure. The most striking and important finding of this unusual study 
(16) is that despite the huge energy expenditure associated with the 2,700 km bike 
expedition, participants did not lose weight. However, such lack of change in body weight 
was completely misleading in terms of energy balance and changes in body composition. 
Indeed, the cyclists lost 2.2 kg of body fat while gaining 2.5 kg of fat-free mass, a 
remarkable finding considering that such changes were observed over a period of only 
two weeks.  
Results of this study provide additional evidence that body weight is a poor outcome to 
assess the effect of a physical activity/exercise program. In our previously published 
exercise training studies, a common observation has been the disconnect between 
changes in body weight and changes in body composition, the latter being sometime 
observed even in the absence of weight loss (10). As weight poorly tracks changes in 
body composition induced by exercise, no wonder why exercise is indeed perceived as 
not being helpful to lose weight by some health professionals. We propose that body 
weight is a misleading outcome when comes the time to evaluate the clinical benefits of 
exercise in individuals with overweight/obese (9).  
One remaining and clinically important issue remains to be addressed regarding the 
effects of a large volume of exercise not inducing weight loss: to what extent substantial 
changes in visceral/ectopic fat could have been observed had imaging data been 
available? In addition, it would have been interesting for the 2,700 km bike intervention 
study to report changes in waist circumference, this metric being more sensitive to regular 
exercise than body weight (12). In our previous intervention studies, it has been our 
experience to find participants losing quite a bit of visceral fat and dropping their waistline 
by several centimeters, while not losing any body weight. 
Thus, on the basis of the above observations, we put forward the hypotheses that: 
1- a very large volume of exercise (1,144 km of bicycle performed for 7 consecutive days) 
will induce major changes in body composition and in levels of visceral adipose 
tissue/ectopic fat in a sample of middle-aged recreational cyclists who will be asked to 
match their energy intake to their very large energy expenditure during the course of the 
one-week extreme exercise intervention. Thus, in a study where, by design, no weight 
loss is planned nor wanted, we expect to see major changes in body composition (slight 
increase in muscle mass and substantial reductions in the size of visceral adipose tissue 
and ectopic fat depots);   
2- changes in levels of visceral adipose tissue and ectopic fat will be better reflected by 
changes in waist circumference – which should decrease in the absence of body weight 
loss. 
We recognize that this is an extreme exercise prescription that has little relevance to public 
health. However, results of this study will be very important in shedding light on the 
importance of shifting our attention from body weight loss to other, more clinically relevant 
fat depots. Thus, the purpose of the study is to provide evidence, through an extreme 
exercise prescription, that weight loss is not the appropriate outcome to evaluate the 
effects of exercise on visceral adiposity and ectopic fat depots (e.g. liver fat and 

4 
 
epi/pericardial fat) which will likely be mobilized to a very significant extent in only one 
week.  
Research Protocol  
A sample of eleven recreational middle-aged male cyclists (aged 50 to 66 years) able to 
perform such a large volume of exercise over a period of one week and asymptomatic for 
CVD will be recruited. The cyclists will be relatively fit with previous participation in 
demanding recreational cycling activities but not retired competitive athletes. From their 
age, we expect them to have subclinical levels of visceral adipose tissue/ectopic fat (these 
depots increase in size with age). 
For this study, participants will be asked to partake in several evaluations: fasting plasma 
lipoprotein-lipid profile and inflammation markers, glycated hemoglobin, cardiorespiratory 
fitness, submaximal exercise test including measurement of energy expenditure, resting 
and exercise blood pressure and heart rate (HR), evaluation of regional adiposity, liver fat 
content, epi/pericardial fat, nutritional quality, and level of physical activity. After baseline 
evaluations, participants will be asked to alternately bike 208 km and 104 km per day on 
a pre-specified course for seven consecutive days. They will be accompanied during each 
of the seven bike rides by research professionals in a recreational vehicle. Participants' 
weight, body composition and waist circumference will be measured under standardized 
conditions in the morning after an overnight fast and after the exercise. Their HR will be 
continuously monitored, and participants will wear accelerometers to estimate their daily 
exercise-related energy expenditure. Foods and fluids will be provided to participants and 
recorded. Again, from the expected large energy expenditure predicted with HR-EE 
equations (between 6,000 and 7,000 kcal per day), participants will be asked to eat at 
least a pre-specified number of calories to be adjusted on the daily basis during the week 
depending upon subjects evolving body weight recorded every morning in standardized 
conditions. At the end of the 1,144 km/7-day bike ride, baseline evaluations will be 
repeated with the exception of the maximal exercise treadmill test, nutritional quality, and 
level of physical activity. To facilitate the conduct of the protocol, the eleven participants 
will be evaluated and followed in two distinct groups.  
METHODS 
A comprehensive set of body fat distribution/ectopic fat variables will be measured on all 
participants as well as lipids and apolipoproteins, cardiorespiratory fitness, submaximal 
exercise test with HR and energy expenditure measured by indirect calorimetry, energy 
expenditure and energy intake evaluation. Questionnaire on nutritional quality and 
physical activity level will also be performed.  
Resting Blood Pressure and Electrocardiogram (ECG) 
Blood pressure measurements will be obtained on all subjects by the same nurse using a 
sphygmomanometer and a stethoscope as previously described (17). Resting blood 
pressure will be recorded in a supine position after a 15-minute rest. A resting 12-lead 
ECG will also be performed. 
Anthropometry and Body Composition 

5 
 
Height, body weight and waist circumference (18) will be measured using standardized 
and previously described procedures. The measurement of body composition will be 
assessed using a bio-impedance device, the InBody 570 (InBody Co., South Korea), 
which provides an analysis of the body per compartment (arms, trunk, and legs). This 
exam is designed to measure body water which in turn allows to measure the lean mass 
and body fat. Bio-impedance will be performed at pre- and post-evaluations as well as 
every morning prior to the 7-day bike rides. 
Fasting Plasma Lipoprotein-Lipid and Apolipoproteins Profiles 
Samples are collected in EDTA tubes in the morning after a 12-hour fast while the 
participants are in a supine position. Cholesterol and triglyceride levels are determined in 
plasma using automated techniques (20, 21). Apolipoprotein (apo) A1 and B levels were 
measured on a Dimension Vista 500 System (Siemens Healthineers, Germany) by 
luminescent oxygen channeling assay on previously frozen plasma samples (-80°C) 
according to the manufacturer’s instructions. 
Abdominal Subcutaneous and Visceral Adipose Tissue 
All imaging will be performed on a Magnetic Resonance Imaging (MRI) 3T system 
(Ingenia; Philips Healthcare, software version R5.1.9, Netherlands) using the integrated 
two-channel body coil for excitation and a 28-channel body array surface coil for signal 
reception. Axial sequence will cover the abdominal region from L2 to S1. Analyses will be 
performed at the level of L4-L5 intervertebral disc.  
Measurement of Liver Fat 
Proton Magnetic Resonance Spectroscopy (MRS) will be performed to determine liver fat 
content (22).  
Measurement of Epicardial and Pericardial Fat 
Levels of epicardial and pericardial fat are both related to features of the metabolic 
syndrome (23-25) and visceral adiposity (25). We therefore intend to measure epicardial 
(covering the epicardium within the pericardium) and pericardial (outside the pericardium) 
fat depots by MRI (26). Epicardial and pericardial fat is routinely measured in the non-
invasive cardiovascular imaging laboratory at the research center. 
Maximal Exercise Test (ETT) 
To assess cardiorespiratory fitness (maximal oxygen consumption, VO2 max), all subjects 
will perform the same symptom-limited maximal exercise test on an Excalibur Sport cycle 
ergometer (Lode B.V., Netherlands) linked to a Cardio2 Ultima gas analyzer system (MGC 
Diagnostics, Minnesota, MI, USA). The incremental protocol consists of 3-min steps with 
increases in the workload of 30 or 35 watts per step. Cardiologists blinded as to LV 
diastolic status will supervise all exercise tests. Blood pressure will be recorded with an 
automatic sphygmomanometer and HR will be recorded continuously during exercise. 
Raw data and average 12-lead ECG will monitor cardiac status every 30 seconds during 
the exercise test as described (27). 
Submaximal Exercise Test (to determine energy expenditure) 

6 
 
The energy expenditure will be estimated using the HR-VO2 relationship. VO2 will be 
estimated by referring to regression lines of HR on VO2 obtained during the measurement 
of resting metabolic rate and the energy cost of submaximal activities such as sitting, 
standing, slow walking, fast walking and cycling at various HRs. Measurements of VO2 
during these activities will be performed using indirect calorimetry. The calculation of 
energy expenditure will be made after stabilization of VO2 and CO2 output, usually during 
the last 2-3 min of each activity as previously described (28). 
Lifestyle Habits Measurements 
Accelerometry (Acti-Graph, Pensacola, FL, USA) recording will be used to average the 
daily energy expenditure at baseline. Sleep time and numbers of steps will also be 
provided by the accelerometer. Accelerometer will be used to record 24-hour HR during 
the 7-day bike ride. HR will be used to extrapolate energy expenditure. 
The quality of diet will be evaluated using the validated Dietary Screening Tool (DST) for 
the assessment of a nutritional quality score which can vary from 0 to 100 (29). Dietary 
habits will be monitored using a validated quantitative web-based food frequency 
questionnaire. During the 7-day bike ride, food and liquid intakes will be detailed, weighed, 
and recorded.  
Finally, all the methodology proposed in the present protocol is well established and 
currently used by the team of investigators. We have all the facilities and equipment to 
conduct the research project as proposed. 
STATISTICAL ANALYSES 
Data will be expressed as mean ± SD. The analysis of the main variables will be the 
differences in changes in visceral adipose tissue and ectopic fat as well as cardiometabolic 
risk markers to document changes following the extreme exercise week. A paired t-test 
will be used with a significance level with a P value below 0.05. 
 
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