Body Composition and Lipid Metabolism at Rest and During Exercise: A Cross-Sectional Analysis.
Conditions: Lipid Metabolism, Substrate Metabolism, Exercise
Sponsor: University of Bath

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STUDY PROTOCOL: 
 
Body Composition and Lipid Metabolism at Rest and During 
Exercise: A Cross-Sectional Analysis 
Version 2 dated 11.12.2018 
INVESTIGATORS 
Dr Javier Gonzalez 
Mr Oliver Chrzanowski-Smith 
 
ADDRESS 
Department for Health 
1 West 4.103 
University of Bath 
Bath, BA2 7AY 
 
Correspondence: J.T Gonzalez@bath.ac.uk 
 

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Overview: 
Obesity and type 2 diabetes (T2D) represent major global public health challenges. In the 
United Kingdom alone, more than 55% of the adult population, are predicted to be obese by 
2050 [1], while the prevalence of T2D is estimated to increase to 9.5% of the population by 
2030 [2]. Moreover, the annual cost of obesity to wider society is estimated to be £27 billion 
in England, rising to nearly £50 billion by 2050 [3]. 
Metabolic inflexibility is proposed to be shared characteristic of obesity and insulin resistance 
[4]. In healthy lean individuals, fat represents the main source of energy during fasting 
conditions, where in response to feeding switches to predominantly glucose (carbohydrate) 
oxidation with fat oxidation supressed; this has been coined ‘metabolic flexibility’ [5]. 
However, individuals with obesity and T2D reportedly do not display normal ‘metabolic 
flexibility’; despite similar rates of fatty acid uptake, skeletal muscle fat oxidation during post-
absorptive resting conditions has been shown to be lower in individuals with obesity and T2D 
compared to lean individuals [6, 7].  
More recently, there has also been a call to further extend the concept of metabolic flexibility 
to encompass a broader range of paradigms such as substrate use during exercise [8, 9]. 
Similarly at rest, fat provides an important source of energy during low-to-moderate intensity 
exercise [10, 11], albeit the contribution of fat is dependent upon numerous factors, including 
nutritional status [12]. During high-intensity exercise however, carbohydrate is the 
predominant fuel source, where an ability to switch between oxidising lipids at lower exercise 
intensities, and carbohydrate at higher exercise intensities, may also reflect an aspect of 
‘metabolic flexibility’. Nevertheless, fat is clearly an important fuel source during fasting and 
prolonged exercise. Furthermore, an impaired ability to utilise fat during such conditions is 
implicated in the pathogenesis of obesity and insulin resistance [5, 7, 8, 13]. 
Correspondingly, lower fasting rates of fat oxidation have been reported in individuals with 
obesity compared to lean individuals [7, 14-17]. Furthermore, greater fasting fat oxidation has 
been associated with lower prospective body weight and fat gain / regain [18-23]. During 
exercise, high rates of fat oxidation have been associated with reduced post-exercise energy 
intake / balance [24-26], greater weight loss maintenance [27], and greater exercise-induced 
fat loss [28]. Importantly, however, this relationship is not consistently observed, with similar 
[14, 17, 29-40] or higher [17, 37, 41-43] rates of fat use at rest and during exercise reported in 
individuals with obesity compared to lean counterparts. Moreover, cross-sectional and 

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prospective associations do not always support the hypothesis that lower fat oxidation is 
associated with greater body weight / fat mass gain or regain [27, 44-47]. Therefore, despite 
being commonly believed, it is currently unclear whether lower fat use at rest or during exercise 
predisposes or is a characteristic of excess adiposity (i.e. obesity). 
The inconsistent cross-sectional case-control and prospective findings between body 
composition and fat oxidation could partly be due to numerous methodological discrepancies 
between studies such as participant characteristics, matching of comparative groups, the 
exercise protocol utilised and / or the assessment of body composition, lipid oxidation and 
cardio-respiratory fitness levels. For example, two recent studies suggest that adiposity may 
not influence fuel use independently of cardiorespiratory fitness in recreationally active males 
[34] and females [33], albeit limited by small sample sizes (n = 24 and 14, respectively) and 
the restricted adiposity range evident (normal and overweight range) [33]. Moreover, adipose 
tissue distribution, not total levels per se, may independently be associated with substrate 
metabolism during exercise [32, 48, 49]. Peak fat use during exercise was reportedly lower in 
females with higher abdominal to lower body fat mass distribution compared to low abdominal: 
lower body fat mass ratio [48, 49]. However, the females recruited were all categorised as being 
of ‘normal’ weight. To our knowledge, no comprehensive data exists on exploring the 
relationships between body composition (total and distribution of body fat), muscle and adipose 
tissue function, and whole-body fat oxidation in both post-absorptive resting and exercising 
conditions, and thus, should be further explored.    
In addition to the potential relationship with body composition, fat oxidation at rest and during 
exercise is known to be influenced by numerous other factors including nutritional status [12, 
50, 51], sex [52-54], exercise mode [53], skeletal muscle fibre composition and glycogen 
content [55] physical activity level (self-reported) [54, 55] and cardiorespiratory fitness [54, 
56, 57]. However, there is reportedly large inter-individual variability in fat oxidation at rest 
[55] and during exercise [54, 55, 58]. While the above factors may partly account for the inter-
individual variability reported, large amounts of variance remains apparently unaccounted for 
e.g. up to 87% in peak fat use during exercise (mg· kg fat free mass-1·min-1) [54]. Furthermore, 
such previous studies have typically recruited small samples of young, healthy active 
individuals, who were also predominantly male, limiting the generalisability of previous 
findings to the wider population. We want to investigate a comprehensive range of novel and 
unexplored variables (e.g. sex hormone concentrations, and skeletal muscle and adipose tissue 
characteristics) alongside previously identified ‘determinants’ of fat oxidation at rest and 

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during exercise in a wide range of adults. This will provide us with a more holistic and broad 
view, allowing determinants to be identified independently of a comprehensive range of 
measured lifestyle, personal, metabolic, genetic and physiological characteristics.  
Lastly, this study will also address the intra-individual variability and reliability of fat oxidation 
at rest and during exercise by asking individuals to complete two identical main trial days 7 – 
14 days apart. Intra-individual variability has become a recent topic of great interest in 
metabolism research [59-61]. The few studies to date that have investigated the intra-individual 
variability of fat use during exercise are not only confounded by the above recruitment issues, 
but have also not reported data on an individual level or systematically applied the wide range 
of reliability statistical methods available [15, 62-68]. Intra-individual variability may partially 
account for the previously reported inter-individual variability in fat use and thus, only once 
accounted for can determinants of fat use be more precisely determined. Importantly, this has 
not previously been considered in determinant studies. Thus, we will apply the entire range of 
reliability statistical methods available to assess intra-individual variability and when reporting 
the results of the current study, will plot anonymous individual data alongside group means. 
This will provide a highly systematic and valid evaluation of the reliability and intra-individual 
variability of fat use at rest and during exercise alongside drawing firmer conclusions on 
potential determinants.  
Overall, this project will extensively explore fat oxidation in a wide range of ‘healthy’ and ‘at 
risk of metabolic disease’ adults under two proposed metabolic flexibility paradigms; post-
absorptive resting and exercising conditions. This will provide important insights into fat use 
at rest and during exercise, help evaluate current and future research, and aide in the design of 
future nutrition and exercise interventions to improve human health. 
 
 
 
 
 
 
 

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Study Aims / Objectives: 
Therefore, the objectives of this study are to comprehensively and systematically explore 
whether whole-body fat use at rest and during exercise is: 
1) Altered in individuals with overweight or obesity compared to lean individuals 
2) Associated with a wide range of physiological, metabolic, lifestyle and genetic 
variables (i.e. potential determinants or health outcomes) 
3) The true inter- and intra-individual variation in fat use which will help to more 
confidently determine the above objectives. 
4) Conduct sub-group analyses to explore any moderators on the association between 
potential determinants and fat use e.g. sex, diet status (e.g. High CHO Vs Low CHO 
dietary intake), menstrual cycle (females only) and sex hormone thresholds. 
Study Design: 
This study proposed is an observational, exploratory cross-sectional study that plans to recruit 
a total of 200 male and female adults aged between 18 – 65 years, who are classed as either 
‘healthy’ or ‘at-risk of metabolic disease’. Participants will visit a laboratory at the University 
of Bath on four occasions. All visits will take place at a Department for Health Laboratory on 
the University of Bath Main Campus. Please see figures 1 and 2 for a study outline and 
schematic, respectively. 
Following advertisement of the study, interested participants will be asked to contact the Chief 
Investigator (Mr Oliver Chrzanowski-Smith [CI]) for further information via email/telephone 
correspondence. If after reading the information sheet, the potential participant is still interested 
in partaking, they will be invited to attend a preliminary meeting at the University of Bath Main 
Campus to further discuss the trial (Visit 1).  
Visit 1 (approx. 45 minutes)  
After the potential prospective participant has read the information sheet, seen the study 
flowchart / schematic outlining the protocol and study demands, had any questions answered 
and are able to competently describe back the study to the CI, they will be asked to sign two 
consent forms (one copy for them, one copy for the CI) if they are still interested in partaking. 
Study eligibility and health screening (via questionnaires) will then be assessed and if eligible, 
a short familiarisation process will take place and a demographic and lifestyle questionnaire 
completed. The familiarisation process involves breathing through a mouthpiece for 5 minutes 

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and 10 minutes of very low intensity cycling (at 30, 40 and 65-Watts). This is in an attempt to 
increase the accuracy of data obtained on each main trial day (Visit 2 and 3). Dates will then 
be scheduled for Visit 2, 3 and 4 and the collection of the preliminary main trial day lifestyle 
measurements (diet and physical activity monitoring). For female participants who are 
eumenorrheic or oligomenorrheic and not using contraception (i.e. combined pill, intrauterine 
device), Visit 2 and 3 will be scheduled to be completed in the same stage of the menstrual 
cycle via the self-reported menstrual cycle questionnaire.  
Visit 2 and 3 (Main Trial Days; approx. 90 minutes) 
Preliminary Main Trial Day Lifestyle Measurements: 
For the 7-days prior to Visit 2 and at least 2-days prior to Visit 3, the participant will be 
instructed to:  
 Wear a physical activity monitor (ActiheartTM) 
 Complete a self-weighed diet diary (this includes at least 3 week-days and at least 1 
weekend day prior to Visit 2, and the immediate 48-hrs prior to the beginning of each 
main trial day) 
 Record step count via a provided pedometer or personal device (e.g. mobile phone app) 
This is to assess participant’s physical activity levels, energy expenditure and energy / 
macronutrient intake. Participants will be asked to replicate their physical activity levels and 
diet 48-hrs prior to the beginning of each main trial day (Visit 2 and 3). Additionally, they will 
be asked to avoid any vigorous physical activity for the prior 48 hrs and avoid alcohol 
consumption for the prior 24 hrs to the beginning of each main trial day. 
Main Trial Days: 
Visits 2 and 3 are identical. For both of the main trial days, participants will be asked to arrive 
at the laboratory after fasting for 12 hrs (± 1 hr). The main trial days can take place either in 
the morning or afternoon, but must be at the same time of the day (± 1 hr) and must take place 
within 7-28 days of each other. The main trial days will follow the below protocol: 
i. Upon arrival to the laboratory, participants will be asked to provide an approximate 
10 mL urine sample and void. 
ii. Anthropometric measurements (body stature (cm), weight (kg), and waist and hip 
circumference (cm)) will then be assessed.  

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iii. After a brief period of rest in a semi-supine position in a resting laboratory, 4 x 5-
min resting expired gas samples will then be collected into Douglas Bags to 
determine resting metabolic rate and substrate use at rest via indirect calorimetry. 
The International Physical Activity Questionnaire (IPAQ) will also be completed 
during the initial brief resting period.  
iv. 
A 10 mL blood sample will then be taken via venepuncture for analysis of 
metabolites and hormones involved in substrate metabolism, and genotyping. 
v. 
After the 10 mL blood sample, the maximal incremental graded cycling test to 
volitional exhaustion (FATMAX test) will be completed (see below for the protocol).  
Once the FATMAX test is finished, the main trial day is complete. If this is the first main trial 
day (Visit 2), participants will then complete the above protocol 7-28 days later. Participants 
will be asked to maintain their habitual lifestyle between Visit 2 and 3.   
Visit 4 (approx. 50 minutes) 
Visit 4 will take place between 2 – 7 days after Visit 3. Participants will be asked to arrive to 
the laboratory after a 12-hr fast at a similar time of the day to visit 2 and 3 (± 1 hr), at least 48-
hrs after Visit 3 and having consumed 568 mL of plain water upon waking. Additionally, the 
participant will be asked to avoid any vigorous physical activity and alcohol consumption for 
the prior 24 hrs to the beginning of this Visit. After voiding, a dual energy X-ray absorptiometry 
(DEXA) scan will be performed to assess body composition. After the scan, the OPTIONAL 
skeletal muscle and / or fat biopsy will then be taken (see below for the skeletal muscle and 
adipose tissue sampling techniques). Once this visit is finished, the participant’s involvement 
in the study is complete. I 
Study Participants: 
We plan to recruit a total of 200 male and female adults who are ‘healthy’ or ‘at risk of 
developing metabolic disease’. Participants will be recruited from within the University of Bath 
and the surrounding areas by email, online and social media platforms (Twitter, University of 
Bath webpage and callforparticipants.com), poster advertising and word of mouth.   
The inclusion criteria is: 
 Male or Female 
 Aged between 18 – 65 years 

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 No current or previous history of known cardio-pulmonary, metabolic or 
musculoskeletal disease  
 A body mass index (body mass in kilograms divided by height in metres squared) 
between 18.9 and 35 kg/m2. 
 All physical activity / fitness levels 
 Willingness and sufficiently able to meet the study demands (including the preliminary 
main trial day measurements and procedures, and maintain their habitual lifestyle 
(physical activity and diet) across the main study period [Visit 1 to Visit 4]).  
Exclusion criteria: 
 Taking any medication that may influence study parameters (lipid and / or 
carbohydrate metabolism) 
 Any reported use of substances, other condition, or behaviour deemed either to pose 
undue personal risk to the participant, or introduce bias into the experiment.  
 Any bleeding disorder or taking any medication which impacts blood coagulation 
 Any female who is breast-feeding or may be / is pregnant 
 Individuals with radio-opaque implants (such as a knee or other joint replacement) or 
medical devices (such as a pacemaker)  
Additional exclusion criteria for individuals who opt in for the optional skeletal muscle and / 
or adipose tissue biopsy are: 
 Known tendency towards keloid scarring 
 Known sensitivity or allergy to any local anaesthetic medicines 
Study Methodology: 
Questionnaires 
Participants will complete several questionnaires throughout the study as outlined under ‘Study 
Design’ (please see the enclosed documents outlining each questionnaire). These consist of: 
a) Health History questionnaire (completed by all participants) 
b) Physical activity readiness questionnaire (PAR-Q; completed by all participants) 
c) Lidocaine Administration Health Questionnaire (to be completed by participants who 
opt for the muscle and / or adipose tissue biopsy) 

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d) Participant Questionnaire (Age, sex, ethnicity, smoking status, dietary patters / special 
dietary requirements; completed by all participants) 
e) Self-reported Menstrual Cycle / Reproductive Status (Females only) 
f) Long Form International Physical Activity Questionnaire (IPAQ; [69])  
Diet Assessment 
In an attempt to standardise upon commencement of trials and analyse potential associations 
with study parameters, participants’ energy and macronutrient intakes will be assessed via a 
self-weighed diet diary in the 7 days prior to Visit 2 and 3. This must include at least 3 week-
days, at least 1 weekend day and the immediate 48-hrs prior to visit 2.  Participants will be 
asked to replicate their diet for 48-hrs prior to each main trial day (i.e. Visit 2 and 3). For Visit 
4 participants will be asked to record their evening. Participants will be provided with a set of 
digital weighing scales (Pro Pocket Scale TOP2KG, Smart Weigh Scales) to accurately weigh 
food and energy-containing drink products. Diet records will be analysed to estimate energy 
and macronutrient intakes (Nutritics Ltd., Dublin, Ireland). These diaries will additionally be 
used to assess study / standardisation compliance. 
Physical Activity / Energy Expenditure Assessment 
Daily physical activity levels (kcal/day and minutes) will be estimated for the 7 days prior to 
Visit 2 and 3 through a combined heart rate and accelerometry chest-worn monitor 
(ActiheartTM; Cambridge Neurotechnology Ltd., Papworth, UK). Individual calibration of the 
monitor from the recorded HR’s in each stage of the respective FATMAX test was subsequently 
performed together with inputting the estimated RMR into the ActiheartTM software to increase 
the accuracy of estimated physical activity energy expenditure [70]. Participants resting 
metabolic rate (kcal/day) will be estimated through indirect calorimetry from the collection of 
the 4 x 5-min resting expired gas samples on each main trial day. The thermic effect of food 
(kcal/day) will be determined by taking 10% of calculated energy intake. Participants physical 
activity level (PAL) will also be expressed as a ratio by dividing total daily energy expenditure 
by resting metabolic rate.  Additionally, participants will be asked to monitor their step count 
for the prior 48 hrs to Visit 2 (via a pedometer or personal device) and approximately replicate 
the number of steps for the 48 hrs prior to Visit 3. The monitoring of steps is to assist in the 
adherence to the standardisation procedures by providing a ‘physical activity marker’ for 
participants. This is in light that ActiheartTM data can only be analysed post-hoc, by which time 

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if physical activity standardisation was not met, would be too late to re-organise the trial, 
potentially invalidating results. 
Collecting and Analysis of Expired Gas Sample (Indirect Calorimetry) 
Expired gas samples will be collected into 100 - 150 L Douglas bags (Cranlea & Hans Rudolph) 
via a mouthpiece connected to a two-way, T shaped non-rebreathing valve (Model 2700, Hans 
Rudolph Inc, Kansas City, Missouri). This valve is connected to a 3-way stopcock valve (Hands 
Rudolph Inc, Kansas City, Missouri) through falconia tubing (Cranlea). Expired gas samples 
will be analysed for concentrations of oxygen and carbon dioxide through paramagnetic and 
infrared transduces, respectively (Mini MP 5200, Servomex Group Ltd., Crowborough, East 
Sussex, UK). The sensors will be calibrated to a two-point low and high calibration (Low: 
99.998% Nitrogen, 0% Oxygen and carbon dioxide; High: Balance nitrogen mix, 20.06% 
oxygen, 8.11% carbon dioxide) using certified, known concentrations (BOC Industrial Gases, 
Linde AG, Munich, Germany). The sensors will be turned on for 30 minutes prior to each trial. 
Ambient oxygen and carbon dioxide concentrations will also be noted during collection of 
expired gas samples to adjust for changes in these concentrations which may have occurred 
due to global climate change and laboratory contamination [71]. Expired gas samples will be 
analysed for at least two minutes and until values are stable. Volume and temperature of expired 
gas samples will be measured using a dry gas meter (Harvard Apparatus) and digital 
thermometer (HI98509 Checktemp® 1, Hanna Instruments Ltd, Bedfordshire, UK), 
respectively, during gas evacuation. Ambient temperature, humidity and barometric pressure 
will be recorded through a weather station (Technoline WS 6730, TechnoTrade Import-Export 
GmbH, Berlin, Germany).  
Substrate use rates will be estimated at rest and during exercise through the stoichiometric 
equations outlined by Jeukendrup and Wallis [72] including adjustment for the contribution of 
glycogen metabolism during exercise.   
Anthropometric Measurements and Body Composition Analysis (DEXA Scan) 
The following anthropometric measurements will be taken: a) Body mass to the nearest 0.1 kg 
using electronic weighing scales (BC-543 Monitor, Tanita, Tokyo, Japan); b) Body stature to 
the nearest 0.1 cm using a wall mounted/attached stadiometer (Holtain Ltd, UK) with 
participants head positioned in the Frankfort plane and after inhalation of a deep breath [73]; 
c) Body mass index (BMI) calculated as mass in kilograms, divided by the square of 
participant’s stature in metres (kg/m2); d) Fat mass index calculated as fat mass in kilograms 

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estimated by DEXA divided by height in metres squared; e) Waist and hip circumference (cm) 
to the nearest 0.1 cm using a measuring tape (SECA 201, Hamburg, Germany) with participants 
arms folded across their thorax. Waist circumference is measured at the narrowest point 
between the 10th Rib and top of the iliac crest at the end of a ‘normal’ expiration [73]. Hip 
circumference is measured at the point of greatest posterior protuberance of the buttocks [73]; 
f) Waist to hip ratio calculated by dividing waist circumference by hip circumference; and g) 
Body fat % to the nearest 0.1% through the bioelectrical impedance analysis function on the 
electronic weighing scales (BIA; BC-543 Monitor, Tanita, Tokyo, Japan). Anthropometric 
measurements will be determined while participants are barefoot and wearing light clothing.  
A Dual Energy X-Ray Absorptiometry (DEXA) scan will be performed at Visit 4 (QDR, 
Discovery W, Hologic, UK). While DEXA was primarily designed to measure bone mineral 
density, a secondary application offered is a non-invasive, precise estimation of total and 
localized body composition. Prior to the scan, the device will be calibrated using a quality 
control material provided by the manufacturer. Participants will be scanned in light clothing 
(i.e. shorts for men and athletic clothing for women). Participants will be positioned centrally 
on the scanning bed; with feet spread apart and hands placed in a mid-prone position such that 
there was a gap between the arms and trunk. This allows regions of interest to be defined by an 
operator using the software provided by the manufacturer (QDR for Windows, Hologic, UK). 
Urine sample analysis 
The 10 mL urine sample will be collected into a 50 mL tube (Corning, Corning Inc, USA) and 
will be analysed for urine specific gravity by a hand-held refractometer (SUR-NE ATEGO, 
ATEGO Co Ltd, Tokyo, Japan). 
Blood sample collection and separation / analysis 
Two x 10 mL whole blood samples will be collected from a vein in the antecubital fossa via 
venepuncture (BD Vacutainer Safety Lok, BD, USA) into a 10 mL syringe (BD Plastipak, BD, 
Madrid, Spain) by qualified and experienced phlebotomists (primarily Dr Gonzalez & Mr 
Chrzanowski-Smith). The blood sample on each main trial day will be split equally into a 5 
mL ethylenediaminetetraacetic acid-coated tube (K3 EDTA, Sarstedt, Germany) and a 10 mL 
serum/clotting activator tube (Serum Z/10 ml, Sarstedt, Germany). The EDTA tube will be 
centrifuged immediately, while the serum tube will be left to stand at room temperature for 20-
min before being centrifuged, both for 15 min at 1700 g (3,000 rpm) and at 4°C (Heraeus 

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Biofuge Primo R, Kendro Laboratory Products Plc., UK). Plasma and serum samples will be 
extracted from the EDTA-treated and non-treated tubes, respectively, by splitting equally 
across eight Eppendorf tubes (4 x plasma, 4 x serum) and immediately frozen at – 20°C, before 
longer-term storage at -80oC for later batch analysis. These samples will be analysed for the 
analysis of metabolites and hormones associated with substrate metabolism (e.g. glucose, non-
esterified fatty acids, triacylglycerol, adrenaline, estrogen).  
The buffy coat layer (consisting of white blood cells) will also be isolated by removing and 
discarding any remaining plasma in the EDTA-treated tube and then carefully pipetting the 
buffy coat layer into an Eppendorf. The buffy coat layer will be immediately frozen at -20°C, 
before longer-term storage at -80oC for later genotyping analysis of single nucleotide 
polymorphisms (SNP’s). SNP’s previously shown to be associated with BMI, waist-
circumference and adipose tissue distribution will be analysed for similar relationships and also 
with substrate metabolism (e.g. FTO [fat mass and obesity associated gene], FFAR1 [long 
chain fatty acid G-coupled protein receptor]). 
Skeletal Muscle Biopsy Procedure (Optional) 
The optional muscle sample (approx. 100 – 200 milligrams) will be obtained from the vastus 
lateralis through the well-established Bergstrom technique at Visit 4. This will be performed 
by trained and experienced personnel only. The area will be thoroughly disinfected and local 
anaesthetic (lidocaine) injected using a small 0.4mm wide, 27G needle. After 5 minutes the 
area will be completely anesthetised and a scalpel will then be used to make a small incision in 
the skin. A needle will then be used to snip some muscle from 2-5 cm beneath the surface of 
the skin and a small stitch will be used to close the incision. Muscle samples will be cleaned, 
processed and frozen immediately for later determination of muscle characteristics related to 
substrate metabolism e.g. the activity and/or content of proteins (fatty acid transporters, 
mitochondria, and enzymes), muscle morphology (fibre type composition, intramyocellular 
lipid content and localisation) and gene expression (mRNA). This will be completed through 
western blots, immunohistochemical and polymerase chain reaction (PCR) analysis 
techniques. 
Adipose Tissue Biopsy Procedure (Optional) 
The optional fat sample (approx. 1-2 grams) will be obtained from the subcutaneous abdominal 
region (approximately 5 cm from the umbilicus) using a well-established needle 

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‘lipoaspiration’ technique at Visit 4. This will be performed by trained and experience 
personnel only. As with the muscle biopsy, the area will first be thoroughly disinfected before 
injection of local anaesthetic (lidocaine) to numb the area. A larger needle will then be inserted 
to collect a small amount of fat tissue (~1g). A syringe will be added to the end of the needle 
to create a slight vacuum, which will help to collect the sample. There will also be some 
movement within the anaesthetised area to dislodge fat cells. As taking the fat sample involves 
using a needle, there is a small risk of infection as with the blood sample via venepuncture, 
however, this risk if minimised by our strict adherence to best practice. Some people experience 
a very small amount of bleeding during the hours immediately after the sample has been taken, 
however you will be closely monitored during the trial day and provided with information 
regarding best practice to looking after the area. In the days following the sampling, you may 
notice some bruising and/or a small lump under the skin, however, these will typically return 
to normal within a few days/weeks, respectively. Adipose tissue will be cleaned, processed and 
frozen immediately for later determination of gene expression (mRNA) and content and 
activity of proteins associated with substrate metabolism. The different types of analysis will 
be performed by PCR, immunohistochemical analysis and western blot techniques.  
 
All biological samples will be stored at -80oC in a freezer(s) located in a locked and secure 
Department for Health laboratory on the University of Bath Main Campus.  
Incremental graded maximal cardiorespiratory cycling exercise test (FATMAX)  
The FATMAX test is an incremental-graded cycling test to volitional exhaustion originally 
designed upon a previously described and validated protocol (GE35/5) in endurance trained 
cyclists [74]. The FATMAX protocol that will be utilised in the current study has also been 
validated in individuals with low cardiorespiratory fitness levels (≥40 mL·min·kg-1 V̇ O2peak) 
[75] and will likely last between 20 – 35 minutes. The FATMAX tests were adapted in an attempt 
to more confidently determine peak fat use and the intensity at which this occurs (FATMAX) in 
individuals with a wide range of cardiorespiratory fitness levels. All tests will be completed on 
a mechanically-braked cycle ergometer (Monark Peak Bike Ergomedic 894E, Varberg, 
Sweden). Figures 3 and 4 depict the two similar FATMAX protocols that will be used.  
The duration of the first seven stages are 4-minutes with approximate 25-watt increments 
(except for the 10-watt first stage increment in protocol 1; see Figures 3 and 4 for exact watt 
increments). From stage 8 onwards, stage duration was shortened to 2-minutes together with 

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greater watt increments between stages (approximate 50-watt increases) until volitional 
exhaustion was reached. The aim of this adjustment was to twofold; 1) To increase the 
sensitivity of the protocol to measure / capture peak fat use in the beginning stages and 2) To 
ensure volitional exhaustion and peak oxygen consumption (V̇ O2peak) were quickly attained 
to reduce the confounding of fatigue. The revolutions per minute (RPM) are 30, 40 and 60 
RPM for the first, second and third stage in FATMAX Protocol 1, and 40 and 60 RPM for the 
first and second stage in FATMAX Protocol 2, where a cadence of 80 RPM is then maintained 
for the remainder of the stages. 
To keep the FATMAX protocols simple and easy to perform for the participant, RPM was 
standardised to multiples of 10 and kept constant where possible. This lead to slight 
discrepancies between the intended and actual watts at each stage (see figures 3 and 4). With 
0.1kg representing the lowest weight available at the University of Bath facilities, the required 
applied mass to achieve the desired watts at each stage was not possible when holding RPM 
constant. Additionally, the weight cage on the Monark Ergomedic 894E peak bike applies a 
constant mass of 1kg, thus to elicit approximately 30, 40 and 65 watts, the RPM at these stages 
were lowered. The two protocols were developed upon a validation study of the FATMAX 
Protocol 2 [75]. In this study participants who had low cardiorespiratory fitness levels often 
reached peak fat use in the first stage (40-watts). Thus, a FATMAX protocol with a first stage of 
30-watts was designed in an attempt to more confidently measure peak fat use in individuals 
with a wide range of cardiorespiratory fitness levels. The FATMAX test protocol to be completed 
by the participant will be decided at Visit 1 after their exercise habits have been verbally and 
written assessed via the health screening and demographic / lifestyle questionnaires. 
Individuals who self-report in engaging in little or no exercise per week / are physically inactive 
will complete FATMAX Protocol 1, whereas regular exercisers will complete FATMAX Protocol 
2. Each individual participant will complete the same FATMAX Test Protocol on the Main Trial 
days (Visit 2 and 3) to ensure comparability.   
To commence the FATMAX Test, participants will be set-up on the cycle ergometer through 
adjustment of saddle and handlebar height and distance to their preferred position. This will 
then be checked and recorded by the researcher to ensure correct and ensure standardisation 
for both tests. Participants will then be explained the respective protocol (See figures 3 and 4) 
and offered the opportunity to ask any questions. A mouthpiece will then be handed to 
participants for 2-minutes in the position expired gas samples will be collected to accustom 

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REC Reference: 17/SW/0269 
them to breathing through the mouthpiece to reduce the likelihood of hyperventilation and try 
to ensure data accuracy.   
Participants will then be instructed to start cycling at the required RPM (either 30 or 40) and 
follow the remaining respective protocol until volitional exhaustion. The stop-watch will be 
started when the RPM of the first stage is reached and stopped immediately upon termination 
of test. Expired gas samples are collected in the final minute (3-4 minute) of the first 7 stages 
and a final expired gas sample (between 30-60 seconds) collected when participants give the 
identified one minute signal to volitional exhaustion.  This final sample allows estimation of 
V̇ O2peak. If a participant continues past this minute, the above process is repeated until 
volitional exhaustion is reached. If the final expired gas sample spans across two stages, the 
intensity will be increased and variance in RPM recorded to appropriately calculate peak power 
output. The mouthpiece is removed in between stages and provided one-minute before each 
collection to ensure adequate ‘flushing’ (excluding final collection). Expired gas samples will 
be analysed immediately after collection with ambient readings returning to stabilised readings 
between time-points before the next bag is analysed. Additionally to ensure stabilised ambient 
gas concentrations are recorded, the Servomex sensor will be left on after analysis of final gas 
sample until environmental readings are stable with any discordant values (i.e. below these 
final readings) adjusted for in the indirect calorimetry calculations. Heart rate (HR) via 
telemetry (Polar RS400 Heart Rate Monitor, Kempele, Finland) and ratings of perceived 
exertion (RPE; on a scale ranging from 6 = no exertion to 20 = maximal exertion [Borg, 1973]) 
will also be recorded during each expired gas sample. Verbal encouragement is provided 
throughout the test and participants are allowed ad-libitum water intake and use of fans. 
Participants will be asked and reminded throughout the test to keep the cadence constant and 
as close as possible to the desired RPM. Consistent or repetitive deviations in RPM (+/- 3) will 
be recorded to appropriately calculate power output.  
Sample Size Estimation 
A sample size estimation of 200 participants was determined via two methods. Firstly, R2 
values (36% and 13%) from two stepwise multiple linear regression models predicting the 
variance of peak fat use during exercise (g/min and mg/kg Fat Free Mass/min, respectively) in 
300 healthy male and female adults were used [54]. Using these R2 values, 42 and 119 
participants were required, respectively, to provide a 95% chance of detecting this variation in 
PFO with α set at 0.05. Secondly, Cohen [76] defines the above effect sizes as large and 

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medium, respectively. A sample size estimation chart produced by Miles and Shevlin [77] with 
ten predictors was consulted as a guide. Conservatively, to detect a medium effect size with a 
power of >95%, approximately 180-190 participants would be required. Taking into account 
the above, a sample of two-hundred participants was decided upon to account for rolling 
recruitment, loss of follow-up data due to study withdrawal, non-completers, replacement of 
drop-outs and any unforeseen issues in data collection. 
 
To minimise the negative impact of loss of follow-up data to validity, participants will be 
provided with a detailed participant information sheet and have the experimental protocol 
explained verbally before the onset of the study. They will be advised to carefully consider the 
required time investment and effort required to complete the study. As such, we hope that 
exclusions based on likely withdrawals can be accounted for prior to participation. Any 
unforeseen change in circumstance or withdrawal of consent resulting in loss of follow-up data 
will be fully documented in the final trial report. 
Statistical / Data Analysis 
Primary Outcome Measure: 
Maximal rate of whole-body fat oxidation (mg/kg 
FFM/min) 
 
 
Secondary Outcome Measures: 
Maximal rate of whole-body fat oxidation (g/min) 
 
FATMAX (% of maximum oxygen consumption) 
 
FATMAX (% of Watt max) 
 
FATMAX (% of Heart Rate max) 
 
Resting rate of whole-body fat oxidation (mg/kg 
FFM/min)  
 
Resting rate of whole-body fat oxidation (g/min) 
 
Resting Metabolic Rate (kcal/min) 
 
V̇ O2peak 
 
Habitual Energy Intake (kcal/day) 
 
Habitual Macronutrient Intake (grams per day) 
 
Habitual Physical Activity Energy Expenditure 
(kcal/day) 

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Menstrual Cycle (Self-reported and sex hormone 
concentrations in blood [Estrogen and Progesterone]) 
 
Fasting measures of blood metabolites: Glucose, 
Triglycerides, Cholesterol, NEFA, glycerol 
 
Fasting measures of Adipokines: Leptin, Adiponectin 
 
Fasting measures of Catecholamines: epinephrine and 
norepinephrine 
 
Fasting measures of sex hormones: 17 beta-estradiol, 
testosterone, progesterone 
 
Fasting pancreatic derived hormone concentrations: 
insulin and glucagon 
 
Hydration Status: Urine Specific Gravity 
 
Age (years) 
 
Sex 
 
Ethnicity 
 
Smoking Status 
 
Medication / Supplement Use 
 
Dietary Patterns / Requirements 
 
Body Fat Percentage 
 
Lean Body Mass (kg) 
 
Body Fat Distribution 
 
Waist and Hip Circumference 
 
Fat Mass Index 
 
Genotype analysis 
 
Skeletal Muscle Characteristics (Protein Content and / 
or Activity) 
 
Adipose Tissue Characteristics (Protein Content and / 
or Activity)  
 
Data will be checked for normal distribution prior to analysis via Shapiro-Wilk’s normality test 
and visual inspection of histograms. Parametric tests will be conducted on normally distributed 
data. Non-normally distributed data will be log-transformed if appropriate or the equivalent 

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non-parametric tests will be conducted. Statistical significance will be set at P < 0.05. The 
statistical analysis for the study objectives identified above are as follows: 
 
1) The effect of body composition on whole-body fat use will be tested by separating 
groups into defined categories (e.g. lean, overweight or obese) and conducting a one-
way ANOVA and mixed design model. The latter to test for any interaction effects 
across the two main trial days.  
2) To identify determinants / characteristics that may be associated with whole-body fat 
use, correlation analysis will firstly be conducted between whole-body fat use at rest 
and during exercise with each variable. Statistically significant correlates will then be 
added to a follow-up hierarchical regression analysis to predict whole-body fat use at 
rest and during exercise.  
3) At a group level, intra-individual variation in whole-body fat use will be tested by a 
paired sample t-test. A range of further reliability / variability tests will be conducted 
to comprehensively determine intra-individual variation: Pearson correlation 
coefficient, Intra Class Correlation, Coefficient of Variation, Bland-Altman plots with 
95% Limits of Agreement and Typical Error. Additionally, individual data will be 
plotted on graphs alongside group means.  
4) The inter-individual variation in fat use will be determined by observing the range of 
whole-body fat use estimates at rest and during exercise recorded after controlling for 
intra-individual variability / calculated measurement error.  
 
Ethical Considerations / Potential Risks and Discomfort 
Skeletal muscle and Adipose Tissue Biopsies: The main ethical issues anticipated relate to the 
relatively invasive optional skeletal muscle and adipose tissue samples to be acquired at Visit 
4. However, as noted, both of these biopsies are optional and thus, participants will be freely 
able to choose whether or not to have these procedures. The skeletal muscle and adipose tissue 
biopsies will be obtained to look at associations between fat use at visit 2 and 3, and 
characteristics of these tissues obtained at Visit 4 (e.g. content and activity of proteins involved 
in substrate metabolism, skeletal muscle fibre composition and mitochondrial content, location 
and function). Both techniques are well established procedures in Human Physiology research 
and are regularly performed in the physiology laboratories at the University of Bath. Only 

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minor complications are typically observed (such as bleeding from the skin wound, bruising 
and minor soreness over the days afterwards). For example, the minor complication rate was 
reportedly 0.15% when using the Bergstrom muscle biopsy technique [78]. Participants will be 
fully explained the risks associated with the procedures and are provided with an extensive 
guide (included with the participant information sheet) to help manage these issues if opted for. 
These sheets also list the possible complications which are more severe (e.g. intramuscular 
bleeding, denervation or infection) although these complications are fortunately very rare and 
risks are further minimised by following best practice. All procedures will be performed only 
by trained individuals to minimize any risk and the minimum amount of each tissue will be 
taken to minimise discomfort whilst gaining the maximum information to answer the research 
questions. 
Anaesthetic: The use of anaesthetic for muscle and adipose tissue biopsies may cause possible 
side effects including allergic reactions, heart arrhythmia and nausea. However, the initial 
health screen will be individually checked by a doctor, who will sign a Patient Specific 
Direction (PSD) to prescribe the anaesthetic for the procedure. A patient information leaflet 
with further information regarding the anaesthetic will also be included in the participant 
information sheet. 
Dual-energy x-ray absorptiometry (DEXA) Scan: A DEXA is a non-invasive technique that 
uses a very low exposure to radiation.  While DEXA was primarily designed to measure bone 
mineral density, a secondary application offered is a precise estimate of total and localized 
body composition. The radiation dose is often compared to the small exposure experienced 
during a short flight (e.g. London to Paris), and a tiny fraction [1/30th] of the amount of 
radiation experienced during a typical chest x-ray. This technique is routinely used in hospitals 
and with elite athletes but nonetheless this does represent some exposure to a small amount of 
radiation. The risks associated with this amount of radiation are described as ‘trivial’ (less than 
1 in a million per whole body scan). This information will be included in the personalised 
feedback provided to participants which would not normally be available to them. The 
exposure of ionising radiation from the DEXA Scan has been reviewed and a declaration made 
by both a lead Medical Physics Expert (Dr Laura Martin) and a lead Clinical Radiation Expert 
(Dr Timothy Jenkinson). 
Cardiac Event: The incremental graded cycling test to volitional exhaustion (FATMAX Test), 
which is performed twice in the study, once at Visit 2 and once at Visit 3, is associated with an 

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increased risk of a cardiac event. The American College of Sports Medicine [79] report an 
incident rate of approximately 6 cardiac events per 10,000 tests. This small risk is likely further 
lowered in individuals with no current or history of cardio-pulmonary or metabolic disease, or 
family history of cardiac events. This test is central to the aims of the study and all sessions 
will be closely supervised by first-aid and CPR trained staff. Participants will have the option 
to freely stop at any stage in the incremental graded cycling tests, further reducing the risk of 
any cardiac event. 
Blood Sampling: Two x approximate 10 mL fasting blood samples across the study will be 
taken via venepuncture (one at Visit 2 and one at Visit 3). These procedures are associated with 
a small risk of infection, embolism or haematoma. Further, these procedures can also cause 
minor bruising and skin irritation. However, the occurrence of such events is very rare and risks 
are further minimised by our strict adherence to best practice.  
Urine Sampling: Two x approximate 10 mL urine samples will be collected across the study 
(one at Visit 2 and one at Visit 3). This will involve the collection of urine in sealed disposable 
containers and storage in a fridge until analysis. The samples will be analysed immediately 
after taken on Visit 2 and Visit 3 with the clinical waste produced disposed as outlined below.  
Clinical Waste: As biological samples are being collected, clinical waste will be produced and 
must be handled appropriately to minimise the risk of injuries and contamination. All 
contaminated equipment (e.g. needles, syringes, urine sample collection containers) will be 
immediately disposed of in clinical waste bins or follow strict adherence to ‘cleansing 
protocols’ outlined in the Standard Operating Procedures developed by the Department for 
Health, University of Bath. The clinical waste bins will be sealed (with autoclave) and collected 
by a member of staff in the Department for Health, University of Bath, before being incinerated 
in accordance with COSHH policies. 
Importantly, risk assessments have been performed and standard operating procedures 
designed by the Department for Health, University of Bath, are in place for all above 
procedures and will be strictly adhered too. 
Time commitment and inconvenience: Participants will be asked to visit the University of 
Bath four times (Visit 1 will take approximately 45 minutes, Visit 2 and 3 will take 
approximately 90 minutes, and Visit 4 will take a maximum of 60 minutes [if both muscle and 
adipose tissue biopsies are opted for]). Testing will take place after fasting for 12-hrs at a time 
in the day most suitable for the participant (subject to availability of research team and lab 

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facilities). Additionally, for lifestyle analysis, participants will be asked to wear a physical 
activity monitor(s) and fill in a self-weighed diet diary during the 7-days before Visit 2 and 
Visit 3. Participants will also be asked to replicate their diet and physical activity for at least 
48-hrs prior to Visit 2 and Visit 3. For some people this may be seen as an inconvenience, 
however, participants will be provided with personalised feedback on their habitual activity 
and diet, which would not normally be available to them. 
Consent Issues: Only persons who are capable of understanding the nature of the trial and what 
it entails will be included in this study (no vulnerable individuals will be included). Even if a 
person gives written consent to take part in this study they will be made fully aware that they 
are free to withdraw from the study at any time without giving any explanation. All participants 
will be fully briefed regarding the nature and risks of all procedures, which are central to the 
research questions, both verbally and in writing before providing consent to take part in the 
study. 
Data and Sampling Handling / Confidentiality: As personal identifiable data is being 
collected there is a small risk to participant confidentiality. This will be addressed by generating 
a unique subject code that will be used on all stored data and samples collected, making it 
completely anonymous. Furthermore, all hardcopies of identifiable information will be stored 
in a locked filing cabinet in a secure office on the University of Bath Main Campus. All 
electronic data / record files will be stored on the University of Bath’s secure server, involving 
a password-protected user log-on and inaccessible to anyone outside of the research team. All 
biological samples will be stored at -80 degrees C in the Department for Health Physiology 
laboratory. This laboratory is alarmed and access it limited to the research group. Any 
published data will be completely anonymised. All personal data and biological samples will 
be destroyed at the end of the study (defined as 5 years after the completion of the final subject, 
by which time all analysis must be finished and any remaining samples destroyed). White blood 
cells from the blood sample (extracted from the buffy coat layer and collected for the isolation 
of DNA), skeletal muscle samples and adipose tissue samples will be stored under the authority 
/ via approval from the NHS HRA South West – Central Bristol Research Ethics Committee. 
y held by ). 
Participant Withdrawal: Participants are freely able to withdraw from the study at any time 
without providing reasons for doing so and without any prejudice. Participants can withdraw 
through contacting any member of the research team. If a participant withdraws one month 

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after their completion of the study, it may not be possible to withdraw their individual data as 
some results may already have been published. This is clearly explained on the participant 
information sheet and as all data will be anonymised, no individual results will be identifiable 
in any way. 
Advertisement for Study Recruitment: Participants will be recruited through advertising the 
study using posters, local press (radio and newspaper advertisements), online social media 
platforms (twitter, facebook, callforparticipants) and the University of Bath website. The poster 
will be displayed throughout the University of Bath and the local area and with appropriate 
permission, in other prominent locations (e.g. local sport and fitness centres, community 
centres, hospitals and GP practices). 
Expenses: Participants will receive a free of charge parking permit for the days they are 
required to visit the University. 
Conflicts of Interests: There are no conflicts of interest for this study. 
Any adverse events relating to the study procedures will be documented and followed up 
until the event is either resolved or adequately explained. 

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0 – 21 days later  
Participants contact researcher 
if interested in partaking 
Visit 1 (approx. 45 mins) 
Preliminary meeting with participant: 

Go through study protocol  

Sign consent forms 

Check study eligibility 

Health screen  

Undergo short familiarisation process  

Organise dates for main trial days and 
collection of activity monitor / diet diary.  
Visit 2 (approx. 90 mins) 
Participant attends lab for first main trial day: 

Fasted state (12 hrs) 

10 mL urine sample and void 

Anthropometric measurements  

Resting expired gas samples and completion of questionnaire 

10 mL blood sample (via venepuncture) 

Complete exercise test 
7 days later 
Collection of activity monitor, diet 
diary and food weighing scales 
Collection of activity monitor, diet 
diary and food weighing scales 
Visit 3 (approx. 90 mins) 
Participant attends lab for second main trial day: 

Replicate physical activity and diet for prior 48 hrs 

Complete identical protocol as highlighted under ‘Visit 2’ Box 
7 days later 

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Figure 1. Study Outline and approx. Timelines  
Within 7 – 14 days 
2 – 7 days 
Provide personalised feedback on 
physical activity, diet and fitness level 
analysis  
End of Main Study 
Visit 4 (maximum 50 mins)  
Participant attends lab for Visit 4: 

Fasted state (12 hrs) 

At least 48 hrs post Visit 3 

No vigorous PA or alcohol in prior 24-hrs 

Consume 568 mL plain water upon waking 

A Dual-Energy X-ray Absorptiometry (DEXA) 
Scan  

OPTIONAL muscle and / or fat tissue biopsy   
Personalised feedback on blood / tissue sampling 
analysis (time received dependent upon when 
analysis begins) 

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Figure 2. Study Schematic 
Recruitment, 
Study Eligibility 
& Familiarisation 
Within        
7 - 28 Days 
Prior 7 
days 
Prior 7 days 
Replicate diet and 
physical activity for 48 
hrs prior to Visit 3 
Visit 3 
Visit 2 
Visit 1 
Visit 4 
Within      
2 – 7 days 
=  Self-weighed 
food diary 
=  Activity 
monitor wear 
= FATmax Test 
=  Urine 
sample 
=  Blood 
sample 
=  Optional 
muscle and / 
or fat biopsy 
=  Anthropometric 
measurements. 
DEXA scan           
(Visit 4 & 5 only) 
=  Resting 
expired gas 
samples 
Key 

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REC Reference: 17/SW/0269 
Figure 3. FATMAX Protocol One 
Figure 4. FATMAX Protocol Two 
 
 
Stage 1 
Stage 2 
Stage 3 
Stage 4 
Stage 5 
Stage 6 
Stage 7 
Stage 8 
Stage 9 
Stage 10 
Stage 11 
Stage 12 
Stage 13 
Stage 14 
Time (mins) 
0 - 4 
4 - 8 
8 – 12 
12 – 16 
16 – 20 
20 – 24 
24 – 28 
28 – 30 
30 - 32 
32 – 34 
34 – 36 
36 – 38 
38 – 40 
40 – 42 
Applied 
Mass (kg) 
1 
1 
1.1 
1.1 
1.5 
1.8 
2.1 
2.7 
3.4 
4.0 
4.6 
5.3 
5.9 
6.6 
RPM 
30 
40 
60 
80 
80 
80 
80 
80 
80 
80 
80 
80 
80 
80 
Proposed 
Watts 
30 
40 
65 
90 
115 
140 
165 
215 
265 
315 
365 
415 
465 
515 
Actual 
Watts 
29.440 
39.254 
64.769 
86.359 
117.762 
141.314 
164.867 
211.972 
266.927 
314.032 
361.137 
416.092 
463.197 
518.153 
Applied 
Mass (kg) 
required for 
proposed 
watts 
1.019 
1.019 
1.104 
1.146 
1.465 
1.783 
2.102 
2.739 
3.375 
4.012 
4.649 
5.286 
5.923 
6.560 
 
Stage 1 
Stage 2 
Stage 3 
Stage 4 
Stage 5 
Stage 6 
Stage 7 
Stage 8 
Stage 9 
Stage 10 
Stage 11 
Stage 12 
Stage 13 
Stage 14 
Time (mins) 
0 - 4 
4 - 8 
8 – 12 
12 – 16 
16 – 20 
20 – 24 
24 – 28 
28 – 30 
30 - 32 
32 – 34 
34 – 36 
36 – 38 
38 – 40 
40 – 42 
Applied 
Mass (kg) 
1 
1.1 
1.1 
1.5 
1.8 
2.1 
2.4 
3.1 
3.7 
4.3 
5.0 
5.6 
6.2 
6.9 
RPM 
40 
60 
80 
80 
80 
80 
80 
80 
80 
80 
80 
80 
80 
80 
Proposed 
Watts 
40 
65 
90 
115 
140 
165 
190 
240 
290 
340 
390 
440 
490 
540 
Actual 
Watts 
39.254 
64.779 
86.359 
117.762 
141.314 
164.867 
188.419 
243.375 
290.480 
337.584 
392.540 
439.645 
486.750 
541.705 
Applied 
Mass (kg) 
required for 
proposed 
watts 
1.019 
1.103 
1.146 
1.464 
1.783 
2.101 
2.420 
3.057 
3.693 
4.330 
4.967 
5.604 
6.241 
6.878 

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