A Randomized Controlled Trial of the Effect of Replacing Sugar-sweetened Beverages With Non-nutritive Sweetened Beverages or Water on Gut Microbiome and Metabolic Outcomes: STOP Sugars NOW Trial
Phase: NA
Conditions: Healthy, Overweight and Obesity, Metabolic Syndrome, PreDiabetes, Diabetes, Overweight, Obesity, Dysglycemia, Cardiovascular Diseases, Cardiovascular Risk Factor, Hypertension
Sponsor: University of Toronto

SIEVENPIPER, John | STOP SUGARS NOW TRIAL |  
Protocol Version 13; updated 17 Aug 2020 
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1. STUDY TITLE  
A randomized controlled trial of the effect of replacing sugar-sweetened beverages with non-nutritive 
sweetened beverages or water on gut microbiome and metabolic outcomes: Strategies To OPpose SUGARS with 
Non-nutritive sweeteners Or Water trial 
2. RUNNING TITLE: STOP SUGARS NOW trial  
3. FUNDING: Canadian Institutes for Health Research (CIHR) 
4. PRINCIPAL INVESTIGATOR   
Prof. John L Sievenpiper, MD, PhD, FRCPC  
Associate Professor, Department of Nutritional Sciences, University of Toronto 
Staff Physician, Division of Endocrinology & Metabolism, St. Michael's Hospital 
Scientist, Li Ka Shing Knowledge Institute, St. Michael's Hospital 
St. Michael's Hospital 
#6137-61 Queen Street East, Toronto, ON, M5C 2T2, CANADA 
Telephone: 416-867-3732 
Mobile: 416-305-4288 
Fax: 416-867-7495 
Email: john.sievenpiper@utoronto.ca  
5. CO-INVESTIGATORS 
Lawrence A. Leiter, MDCM, FRCPC 
Division of Endocrinology and Metabolism 
St. Michael’s Hospital 
#6121-61 Queen Street East, Toronto ON, M5C 2T2, Canada 
Telephone: 416-867-7441 
Email: leiterl@smh.ca  
6. POST-DOCTORAL FELLOW 
Tauseef Khan, PhD 
St. Michael's Hospital 
61 Queen Street East 
Toronto, ON, M5C 2T2, CANADA 
Email: tauseef.khan@utoronto.ca 
 
GRADUATE STUDENTS 
Néma McGlynn, RD 
St. Michael's Hospital 
61 Queen Street East 
Toronto, ON, M5C 2T2, CANADA 
Email: nema.mcglynn@mail.utoronto.ca  
Sabrina Ayoub-Charette 
St. Michael's Hospital 
61 Queen Street East 
Toronto, ON, M5C 2T2, CANADA 
Email: sabrina.ayoubcharette@mail.utoronto.ca 
 

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7. BACKGROUND AND SIGNIFICANCE  
International health agencies [1] and chronic disease associations [2, 3] have called for reductions in free/added 
sugars to ≤5-10% of energy to address the growing epidemics of obesity and diabetes. Attention has focused 
especially on the reduction of the major source of free sugars, sugar sweetened beverages (SSBs), the excess 
consumption of which has been associated with weight gain, diabetes, and their downstream complications 
including hypertension, and coronary heart disease (CHD) [4-7]. Ontario’s Healthy Kids Panel [8], Health Canada 
[9, 10], the Standing Senate Committee [11], the Heart and Stroke Foundation [2] and Diabetes Canada [3] have 
recommended policies to reduce SSBs including replacement strategies, taxation, and/or bans on advertising to 
children. A role for non-nutritive sweeteners (NNSs) in these policy options has been conspicuously absent.  
There is an emerging concern that NNSs may contribute to an increase in the diseases that they are trying to 
prevent. Systematic reviews and meta-analyses of prospective cohort studies have shown that NNSs are 
associated with increased risk of weight gain, diabetes, and CHD [5, 12]. Although this evidence is recognized to 
be at high risk of reverse causality and disagrees with the higher quality evidence from randomized controlled 
trials [13], several biological mechanisms have been proposed, among them changes in gut microbiome. One 
highly-influential study [14] concluded that NNSs induce glucose intolerance through a loss of diversity in 
microbiome. This study, however, disagreed with a subsequent study [15] and had several methodological 
weaknesses including the lack of a control group. Despite the uncertainties, these data have contributed to a 
negative view of NNSs in the media [16-23].   
There is an urgent need to address the ongoing concerns related to NNSs. Health Canada, in particular, has 
indicated that studies of sugar reduction strategies that use NNSs and target microbiome are an important 
research priority [24]. We propose to conduct a CIHR-funded randomized controlled trial that assesses the effect 
of a ‘real world’ strategy to reduce SSBs using non-nutritive sweetened beverages (NSBs) or water on gut 
microbiome, glucose tolerance, and cardiometabolic risk factors in overweight or obese participants. 
 
8. OBJECTIVES  
1. To assess the effect of replacing SSBs with NSBs or water on one primary outcome of diversity of gut 
microbiome over 4-weeks in overweight or obese participants. 
2. To assess the effect of replacing SSBs with NSBs or water on another primary outcome of glucose 
tolerance over 4-weeks in overweight or obese participants. 
3. To assess the effect of replacing SSBs with NSBs or water on the secondary and exploratory outcomes of 
body weight, blood pressure, glucose and insulin regulation, blood lipids, ectopic fat, liver fat, body 
adiposity, and diet quality over 4-weeks in overweight or obese participants 
 
9. HYPOTHESIS 
1. Replacing SSBs with NSBs or water will change the diversity of gut microbiome after four weeks in 
regular SSB drinkers. 
2. Replacing SSBs with NSBs or water will increase glucose tolerance after four weeks in regular SSB 
drinkers. 
3. Replacing SSBs with NSBs or water will decrease body weight, reduce blood pressure, improve glucose 
and insulin regulation, decrease blood lipids, decrease ectopic fat, decrease liver fat, decrease body 
adiposity, and improve diet quality after four weeks in regular SSB drinkers.  
 
10. METHODS 
10.01 Trial Design. We will conduct a single-centre, open label, randomized controlled crossover trial with three 
four-week intervention phases (SSB, NSB, water) comparing the effect of replacing SSBs with NSBs or water on 
the gut microbiome and glucose tolerance. A cross-over design was selected due to the high inter-individual 
heterogeneity in gut microbiome [25]. Prior to randomization, participants will undergo a ≥2-week run-in phase 

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to gather data on SSB consumption prior to study start. Each participant will then act as their own control 
receiving the 3 interventions in random order, each separated by a ≥4-week wash-out phase (Figure 1). We will 
collect fecal samples from participants and conduct Oral Glucose Tolerance Tests (OGTTs) before the start and at 
the end of each intervention for gut microbiome analysis and glucose tolerance.  All study visits will occur in 
Toronto at the Clinical Nutrition and Risk Factor Modification Centre at St. Michael’s Hospital. The microbiome 
analysis and the objective biomarkers of SSBs assays will be performed at the Department of Nutritional 
Sciences, University of Toronto; plasma glucose analysis will be performed at the Banting and Best Diabetes 
Center (BBDC), University of Toronto; plasma insulin analysis and other blood analyses (for a selected sample 
using a multiplex kit for adiponectin, adipsin, C-Peptide, Ghrelin, GIP, GLP-1, Glucagon, Insulin, leptin, PAI-1, 
Resistin and Visfatin) will be conducted at the Guelph Research and Development Centre, Ontario, Canada. Lipid 
analyses will be performed at St. Michael’s Hospital Core Lab.  
 
Figure 1: Overview of the study visits and phase.*  
 
 
 
 
 
 
 
*All possible groups are not shown.  
 
10.02 Blinding. Blinding of the participants and investigators will not be possible due to packaging, taste and 
look of the interventions. However, outcome assessors (laboratory, microbiome analysis) and the statistician will 
be blinded to the identity of the treatments. 
 
10.03 Participants. We will recruit 81 overweight or obese (ethnic specific cut-offs for overweight and obesity 
are used ≥23 kg/m2 for Asian individuals and ≥25 kg/m2 other individuals[11]), adult (age, 18-75 years) men and 
non-pregnant women (with no conception plans for the study duration) with a high waist circumference (≥94cm 
in men, ≥80cm in women in Europid, Sub-Saharan African, Eastern Mediterranean, and Middle Eastern 
individuals; ≥90cm in men and ≥80 cm in women for South Asian, Chinese, Japanese, and South and Central 
American individuals[26]) who are otherwise healthy (free of disease or illness), not taking medications that 
have a clinically relevant effect on the primary outcomes, not smoking, have no intentions of making major 
alterations to their diet or physical activity regime during study duration, and consuming ≥1 servings of SSBs per 
day (for which there is an NSB equivalent that contains the non-nutritive sweeteners acesulfame potassium or 
sucralose). Participants must be agreeable to consume only one type of SSB and brand-matched NSB for each 
month of intervention.  If participants are consuming NSBs in addition to SSBs they must be willing to forgo them 
for the run-in and duration of the study. All participants must have a primary care physician and be proficient in 
English to be eligible for the study. Please see Table 1 for a full set of exclusion criteria. SSBs will be defined as 
any sugar-sweetened beverage (sodas and soft drinks) that contains at least 50 kcal per 8-oz serving. For the 
purpose of the study, SSBs will NOT include fruit drinks, sports/energy drinks, sweetened iced tea, coffee, or 
homemade SSBs such as frescas or 100% fruit juice. All participants will be expected to participate in the study 
for approximately six months (one run-in phase of two weeks, three intervention phases of four weeks each, and 
two wash-out phases of four weeks each) and be able to attend all study visits. They must consent to have the 
164 Days (5.5 months) 
Analysis 
1
5 
3
2 
  6 
4 
Group 1
Group 2 
Group 3 
SSB 
NSB 
Water 
Wash out 
Wash out 
Wash out 
SSB 
NSB 
Water 
Wash out 
Wash out 
Wash out 
SSB 
NSB 
Water 
Randomizatio
4 weeks 
≥4 weeks 
4 weeks 
≥4 weeks 
4 weeks 
0 
Visits 
≥2 weeks 

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study drinks shipped to their home address by a third-party company, who will not be informed of their 
participation in the study, or be agreeable to pick up study drinks from the study site at St. Michael’s Hospital. 
 
Of the 81 participants, 30 of them will be asked to consent to have an MRI taken to measure their liver and 
muscle adiposity.  
Table 1 – Study exclusion criteria* 
Age <18 or >75 years 
BMI <23 kg/m2 for Asian individuals and <25 kg/m2 other individuals 
Waist circumference <94cm in men, <80cm in women in Europid, Sub-Saharan African, Eastern Mediterranean, 
and Middle Eastern individuals; <90cm in men and <80 cm in women for South Asian, Chinese, Japanese, and 
South and Central American individuals 
Not regularly drinking SSBs (≥1 serving per day) 
Self-reported pregnant or breast feeding females, or women planning on becoming pregnant throughout study 
duration  
Regular medication use that have a clinically relevant effect on the primary outcomes (except birth control and 
PRN meds such as Advil, Tylenol, etc) 
Antibiotic use in the last 3 months 
Complementary or alternative medicine (CAM) use as deemed inappropriate by investigators 
Self-reported diabetes 
Self-reported uncontrolled hypertension (or systolic blood pressure (BP) ≥ 160 mmHg or diastolic BP ≥ 100 
mmHg [27]) 
Self-reported polycystic ovarian syndrome 
Self-reported cardiovascular disease 
Self-reported gastrointestinal disease 
Previous bariatric surgery 
Self-reported liver disease  
Self-reported uncontrolled hyperthyroidism or hypothyroidism 
Self-reported kidney disease 
Self-reported chronic infection 
Self-reported lung disease 
Self-reported cancer/malignancy 
Self-reported schizophrenia spectrum and other psychotic disorders, bipolar and related disorders, and 
dissociative disorders 
Major surgery in the last 6 months 
Other major illness or health-related incidence within the last 6 months  
Smoker 
Regular recreational drug users  
Heavy alcohol use (>3 drinks/day) 
Do not have a primary care physician 
Participation in any trials within the last 6 months or for the duration of this study 
Any condition or circumstance which would prevent you from having an MRI (e.g. having prostheses or metal 
implants, tattoos, or claustrophobia) (this is applicable for the 30 participants in the MRI portion of the study) 
Individuals planning on making dietary or physical activity changes throughout study duration 
*Disease exclusions will be based upon self-reported diagnosis  
10.04 Sample Size (Power) Calculation.  
The study will be performed in a total of 81 participants. It is powered to show a difference between the water, 
NSB, and SSB arms in 60 participants in the two primary outcomes. Assuming a drop-out rate of 20 percent, we 

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would need 75 participants in order to have to power to detect a difference. Since our final recruitment strategy 
resulted in a large number of participants who made it through the run-in phase, we decided to increase our 
recruitment in order to replace drop outs and thus will recruit 81 participants.   
 
One primary outcome is in beta diversity of the gut microbiome communities of the participants between water 
and NSB groups via 16S ribosomal rRNA gene sequencing. We used the micropower R package to compute 
sample size based upon the power of 16S tag sequencing that can be analyzed using pairwise weighted UniFrac 
distances [28]. UniFrac is a distance metric based upon the unique fraction of branch length in a phylogenetic 
tree built from two sets of taxa. Comparison of microbiome samples is performed via weighted UniFrac, which 
considers the relative abundance of taxa. We simulated the within-group distance as 0.2, and the standard 
deviation (SD) of within-group distances as 0.07. To detect a weighted UniFrac distance of 0.04, which is smaller 
than the effect observed in a studies of Suez et al. [29] (0.05 derived from figure 5), and considering it is a 
cross-over study with a within-person correlation of 0.7, and taking into account multiple arms we calculated 
that for above 95 percent power we would need 60 participants in this study. Assuming a loss of 20 percent, we 
will recruit 75 participants. We are confident about detecting an alteration in gut microbiome diversity if it exists 
as previous studies show that small changes in diet causes significant alteration in gut microbiome taxa over a 
much shorter period (5 to 7 days) in fewer individuals (10 to 25 people) [25, 30].  
 
Another primary outcome is glucose tolerance, as measured by incremental Area Under the Curve (iAUC) from a 
2-hour 75g OGTT. With 60 participants we will have 89 percent power (assuming absolute numbers for mean 
and SD from our recent unpublished randomized trial) to detect a 20% change in mean iAUC between the water 
and NSB group  if the direction of change is similar to Suez et al. [29] while assuming a within-person correlation 
of 0.7 and taking into account the three comparisons. The 20% difference for glucose iAUC is based on the 
minimally important difference proposed by Health Canada to support postprandial blood glucose response 
reduction claims [31]. 
This power calculation takes into account adjustment for multiple testing for both primary outcomes using the 
Benjamini-Hochberg procedure, which is a suggested method by the Food and Drug Administration in its 
“Multiple Endpoints in Clinical Trials Guidance for Industry” [32]. Benjamini-Hochberg procedure is a step-down 
method that controls for false discovery rate, while maintaining high power [33, 34].  We will implement a 
truncated Benjamini-Hochberg method with parallel gatekeeping in which some portion of the unused alpha 
from each step is reserved for passing to the secondary outcome family if any of the primary outcome is 
significant. The alpha levels calculated for the primary outcomes is given in table 2. The study is also powered to 
show a difference between the three arms for secondary outcomes with an α=0.0125, the lowest possible 
starting α for secondary outcomes based upon the truncated Benjamini-Hochberg procedure.  
Sub-study: 1H-MRS will be performed in 25 subjects to assess intrahepatic and intramuscular fat. We will have 
99% to show a difference in liver fat of 5% between the water and NSB arm for both hepatic and muscle fat 
assuming a between group SD of 4%, with a correlation of 0.65 and alpha of 0.05.  
Table 2. Power calculation for primary outcomes between water and NSB arms 
Outcome type 
Outcome 
Mean 
change 
SD 
Correlation 
N 
Alpha* 
Power (Beta) 
N (corrected for 20% 
loss to follow up) 
Primary outcomes family 

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Primary  
Microbiome 
UniFrac Distance 
0.04 
0.07 
0.7 
60 
0.0375 
98% 
75 
Primary 
Glucose iAUC 
44.81 
mmol/L/min 
(20%) 
113 
mmol/L/min 
0.7 
60 
0.025 
89% 
75 
  P-value order will be from largest to smallest.  
10.05 Recruitment. Participants will be recruited through postings and flyer handouts at St. Michael’s Hospital, 
the University of Toronto, local clinics, and surrounding areas, advertisements in newspapers and on transit, and 
online listings on Craigslist and Kijiji. Some participants will complete a Survey Monkey to assess general 
eligibility. We will be utilizing a digital marketing group, TrialFacts, to launch a campaign to recruit participants. 
TrialFacts works exclusively with research groups to provide specialized patient recruitment services for clinical 
trials. Participants will be screened first by telephone. Eligible participants will be called in for an information 
session on the study including being provided the consent form. Those that wish to participate will provide 
written consent and will subsequently have an in-person screening including blood work. Based on our most 
recent FACE trial (NCT02459834), we estimate a recruitment rate of 1-2 participants/week and therefore plan to 
recruit all the participants within a 6-month period. Dr. Tauseef Khan will organize the recruitment. Our MSc 
candidate #1 (Néma McGlynn, RD) and our Research Coordinator  will conduct the screening. 
10.06 Screening and Consent. Interested individuals will call the study coordinator and be screened over the 
phone for eligibility using a questionnaire and be briefed on the nature of the study, treatments, and 
procedures. Individuals who qualify and are interested will be asked to come to the Clinical Nutrition and Risk 
Factor Modification Centre at the St. Michael’s Hospital (study site) for an information session where they will 
be given the consent form. They will be permitted to take the form home so that ample time to review study 
procedures. Participants who provide written consent will be assigned a screening identification (ID) and will be 
scheduled for an in-person screening will be arrange to obtain anthropometric measures as well as 
questionnaire administration regarding SSB intake to confirm study qualification. The anthropometrics will 
include height, weight, waist circumference, and blood pressure. Automated Office Blood Pressure (AOBP) 
measurements at the first screening visit will be recorded in both arms unless a physical condition necessitates 
the use of a particular arm. The arm with the highest mean systolic measurements at screening will then be used 
for all subsequent AOBP assessment throughout the study. If the mean of 3 AOBP measurements in the arm 
with the highest systolic measurements shows systolic blood pressure (SBP) >=160 mmHg or diastolic BP (DBP) 
>=100 mmHg, then the patient will be excluded for uncontrolled hypertension [27].     
Any significant results will be provided to participant’s family physician for further follow-up. Those who meet all 
the eligibility criteria will be enrolled in the run-in phase of the study, which will last two weeks, and then be 
assigned a study ID. Participants will be instructed on details of the trial protocol. 
10.07 Run-in Phase. Participants who are eligible following in-person screening will also start the ≥2-week run-
in phase. For two weeks during the run-in phase and prior to the first study visit, participants will complete a 
beverage log sheet. They will also be instructed on how to collect a 24-hour urine sample and fecal sample and 
provided kits to do so for the first study visit. The participants will be provided with their beverage log sheets for 
the month intervention. During the run-in phase participants will begin to log their usual beverage intake as well 
as asked for their beverage preference of SSB and NSB.  
10.08 Randomization. Using the Research Electronic Data Capture (REDCap) program, the Applied Health 
Research Centre (AHRC) will perform the randomization with no stratification. Following successful completion 
of the run-in phase and following measurements taken at the first study visit, participants will be randomized 
into groups, of a possible six, using a blocked (Latin squares) randomization. These groups will be sequences 

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representing SSB, NSB and water groups. The Latin square sequences will be randomly allocated to participants 
with a similar number of participants allocated to each treatment sequence (Table 2). The randomization 
schedule is also created by AHRC through REDCap. The participants will only be randomized and given their 
study drinks once all measures from the first study visit are collected. 
Table 2: Latin square randomization for three group cross-over study.  
 
Intervention Phase 1 
Intervention Phase 2 
Intervention Phase 3 
 Sequence Group 1 
A 
B 
C 
 Sequence Group 2 
B 
C 
A 
 Sequence Group 3 
C 
A 
B 
 Sequence Group 4 
B 
A 
C 
 Sequence Group 5 
A 
C 
B 
 Sequence Group 6 
C 
B 
A 
Rows represent all possible sequences and columns represent different intervention phase. 
 
10.09 Intervention. There will be three interventions: Participants will be provided with the SSB of their choice 
from Table 3 (355 ml, 140kcal, 42g sugars per can), equivalent NSB (355 ml, 0kcal, 0g sugars per can), or water 
(355 ml, 0kcal, 0g sugars per can or bottle of still or carbonated water) to replace the amount of SSBs they 
usually consume (≥1serving/day) as determined in the run-in phase. All intervention beverages will be provided 
to each participant. They will be instructed to replace their usual SSBs with the study beverages while freely 
consuming their usual background diets. The calories of the intervention groups will not be matched to allow for 
“real-world” substitutions using products available on the market. They will pick up one week of their beverage 
assignment at the first visit of each phase and then will have the remaining three weeks of beverages delivered 
using an online grocery delivery service (i.e. Grocery Gateway (www.grocerygateway.com)). Our MSc candidate 
#1 (Néma McGlynn, RD) and our research coordinator  will coordinate the beverage disbursements. The 
participants will receive relevant drinks during the intervention phase based on their group assignment. They 
will revert to their usual SSB intake during wash-out phase during which they will not receive any beverages 
from the study. 
Table 3: A full list of the three interventions and available beverages is given below.  
 
 
 
SSBs group – 
355 ml can 
42 grams of sugars 
NSBs group – 
355 ml can 
0 grams of sugar 
Water group –  
355 ml bottle 
or can 
Coca-Cola  
 
Pepsi 
 
 
Canada Dry Ginger Ale 
Schweppes Ginger Ale 
Mountain Dew 
Sprite 
7UP 
Orange Crush  
Diet Coke (Aspartame, Acesulfame) 
Coca Cola Zero (Aspartame, Acesulfame) 
Diet Pepsi (Aspartame, Acesulfame) 
 
Diet Canada Dry Ginger Ale (Aspartame, Acesulfame) 
Diet Schweppes Ginger Ale (Acesulfame, Sucralose) 
Diet Mountain Dew (Aspartame, Acesulfame, Sucralose) 
Sprite Zero (Aspartame, Acesulfame) 
Diet 7UP (Aspartame, Acesulfame) 
Diet Orange Crush (Sucralose) 
 

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10.10 Study visits (Week-0 and Week-4 for all 3 interventions, 6 total study visits):  
The trial will be conducted at the CFI-funded Toronto 3D Clinical Research Centre and MRI Research Unit and at 
the Clinical Nutrition and Risk Factor Modification Centre at St. Michael’s Hospital. Each participant will receive 
the three interventions in random order, each separated by a ≥4-week washout. Participants will receive 
motivational phone calls every 2-weeks between visits to provide reminders and ensure fidelity to the protocol.  
For Week-0 of first intervention only: The first visit will be scheduled upon finalizing the run-in phase. 
Participants will come to the study site and their beverage logs will be checked for completion for the 2-week 
run-in phase, any changes required will be communicated. Participants will be notified of their randomization 
assignments after completing all of their baseline testing.  
For all six study visits: Participants will come to the study site prior to their visit to pick up standard stool and 
urine collection kits as well as be instructed on how to complete a three-day diet record (3DDR) using Nutritics, a 
nutrition software available for use on smartphones (a paper form of the 3DDR will be available for participants 
who do not have access to a smartphone or prefer not to use the application). Participants will be provided with 
instructions for stool collection, storage, and delivery to the study centre. Stool samples will be collected in 
specimen containers surrounded by dry ice packs, and brought to the centre by the participants and stored in a -
80C freezer. At each visit, study staff will retrieve the stool sample, the 24-hour urine collection, and beverage 
logs. Subjects will come in to the study site in a 10 to 12-hour fasted state. They will be instructed to maintain 
the same dietary and exercise patterns the evening before each test and consume a minimum of 150g of 
carbohydrate each day over the three days prior to the test.  To ensure that these instructions are followed, 
participants will be provided with examples of what constitutes 150g of carbohydrate. Their weight, waist 
circumference, and blood pressure will be measures. A fasting blood sample for primary, secondary, and 
exploratory outcomes (including glucose, insulin, lipid panel, ALT and other liver enzymes, and other markers of 
glucose control, biomarkers of adherence, and other biomarkers of cardiometabolic risk) will be taken by an 
intravenous nurse. A standard protocol will be followed for the administration of a 2-hour 75g-OGTT. 
Participants will have a catheter inserted into a forearm vein by the nurse, secured by tape, and kept patent by 
saline. Two baseline samples will be obtained in the fasting state at 0-min. A 75g OGTT meal (GlucoDex 75g, 
Rougier Pharma Mirabel, QC, Canada) will then be administered with instructions to consume it over exactly 5 
min at a constant rate using a timer. Additional blood samples will be drawn at 30, 60, 90, and 120 min after the 
start of the test, at the end of the test the nurse will remove the catheter.  Breakfast will be prepared at the 
study site kitchen and provided to the participants after the OGTT. Staff will also administer case report forms 
and questionnaires which will include information on diet (cravings, hunger and fullness), physical activity, 

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medication changes, and symptoms. The study dietitians will review the 3DDR with participants. For the 30 
participants who will participate in the imaging portion of the study, a 1H-MRS scans will be conducted in the 
CFI-funded Toronto 3D Centre MRI research unit at St. Michael’s Hospital. The 1H-MRS will be performed to 
measure ectopic fat in liver and calf muscles (Table 4). Once all measures have been collected, the participant 
will be provided the intervention. For week-0 of all 3 interventions, participants will be given 1 week’s worth of 
beverages to take home; the beverages for the remaining 3 weeks will be delivered using an online delivery 
service (Starhawk express (https://www.starhawkexpress.com/)). They will also be provided with their beverage 
log sheets for the month 
Washout (2 x 4 weeks):  
At the end of week-4 for the first two interventions, the wash-out phase of ≥4 weeks will start for which 
participants will be asked to revert back to their usual beverage intake. Participants will be given a beverage log 
to complete over this four-week phase. No beverages will be provided by the study site during the wash-out 
phase. The study coordinator will call the participants near the middle and near the end of each phase to remind 
them of their group assignment and the next study visit. Reminders will also be sent via email. 
Table 4: Participant Visit Schedule  
 
 
10.11 Outcomes 
The two primary outcomes are change in gut microbiome beta diversity, measured by 16S rRNA gene 
sequencing, and plasma glucose iAUC, measured by OGTT.  
Secondary outcomes are change in waist circumference, body weight, fasting plasma glucose, 2h plasma glucose 
[2h-PG], and the Matsuda whole body insulin sensitivity index [Matsuda ISIOGTT] [35].  
Phase (Length in weeks) 
Screening 
(0) 
Run-in 
(2 weeks) 
Intervention 
phase 1 
(4 weeks) 
Wash-out 
(4 weeks) 
Intervention phase 
2 
(4 weeks) 
Wash-out 
(4 weeks) 
Intervention phase 
3 
(4 weeks) 
Study Visit 
0 
No visit 
Week 0  
(Start) 
Week 4 
(End) 
No visit 
Week 0 
(Start) 
Week 4 
(End) 
No visit 
Week 0 
(Start) 
Week 4 
(End) 
Written Informed Consent 
Provided 
x 
 
 
 
 
 
 
 
 
 
Anthropometric measures 
Height 
x 
 
 
 
 
 
 
 
 
 
Weight  
x 
 
x 
x 
 
x 
x 
 
x 
x 
Waist circumference 
x 
 
x 
x 
 
x 
x 
 
x 
x 
Blood pressure 
x 
 
x 
x 
 
x 
x 
 
x 
x 
Biochemical measures 
 
 
 
 
 
 
 
 
 
 
 
Fecal sample 
 
 
x 
x 
 
x 
x 
 
x 
x 
24-hour urine collection 
(biomarkers for compliance) 
 
 
x 
x 
 
x 
x 
 
x 
x 
Blood sample drawn 
 
 
x 
x 
 
x 
x 
 
x 
x 
OGTT 
 
 
x 
x 
 
x 
x 
 
x 
x 
Questionnaires 
Personal information 
x 
 
 
 
 
 
 
 
 
 
Demographic Data 
x 
 
 
 
 
 
 
 
 
 
Medical History 
x 
 
 
 
 
 
 
 
 
 
Case Report Form (eg. 
medications, physical activity) 
x 
 
x 
x 
 
x 
x 
 
x 
x 
Beverage log 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Symptoms questionnaire 
 
 
x 
x 
 
x 
x 
 
x 
x 
Three-day diet record 
 
 
x 
x 
 
 
x 
 
 
x 
Cravings/hunger/satiety 
questionnaires 
 
 
x 
x 
 
x 
x 
 
x 
x 
Radiology 
Liver fat (MRS) 
 
 
x 
x 
 
x 
x 
 
x 
x 

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Exploratory outcomes are change in blood pressure; fasting plasma insulin; 75g OGTT derived indices (iAUC 
plasma insulin, maximum concentrations (Cmax) and time to maximum concentrations (Tmax) of plasma glucose 
and insulin, mean incremental plasma glucose and insulin, homeostatic model assessment of insulin resistance 
(HOMA IR) and the insulin secretion-sensitivity index-2 [ISSI-2]); cardiometabolic risk (lipid panel) ectopic fat (an 
early metabolic lesion) in liver (intra-hepatocellular lipid [IHCL]) and calf muscles (intra-myocellular lipid [IMCL]) 
by 1H-MRS; fasting blood lipid profile; satiety, hunger, and food cravings (using the Control of Eating 
Questionnaire), and diet quality (by analysis of the 3DDRs)[36].  
Adherence outcomes will be based on participant beverage logs, returned beverage containers, and objective 
biomarkers of SSBs (increased 13C/12C ratios in serum fatty acids, increased urinary fructose), water (decreased 
13C/12C ratios in serum fatty acids, decreased urinary fructose), and NSBs (urinary acesulfame potassium, 
sucralose) intake. 
10.12 Compliance assessment 
To assess compliance, participant beverage logs will be checked at each visit. In addition, participants will return 
unused SSBs, NSBs, or water bottles to assess compliance. Urinary analysis will be obtained for biomarker 
analysis for compliance (Biomarkers of Adherence, page 9). 
10.13 Analytical Techniques 
Anthropometric analyses.  
Height will be measured with a wall-mounted stadiometer (Perspective Enterprises, Portage, MI, USA).  
Body weight will be assessed by beam scale.  
Waist circumference will be assessed using the Heart and Stroke Foundation methodology [37].  
Blood pressure (BP) and resting heart rate will be measured. To collect this measure, participants will remain 
seated in a quiet, temperature-controlled room for at least 5 minutes to achieve resting heart rate and BP. 
Subsequently, BP will be measured oscillometrically using the OMRON Intellisense HEM-907 according to JNC VII 
criteria. BP will be measured in triplicate, with each measurement separated by one minute, and the average of 
the three measures will be taken [38].  
Biochemical analyses 
Fecal microbiome analyses. DNA will be extracted from fecal samples and compositional analysis will be done 
by next generation sequencing. Primers will be used to target the appropriate regions of the 16S rRNA gene for 
pair-end sequencing using Illumina MiSeq [39]. Sequencing data will be analyzed to assign Operational 
Taxonomic Units (OTUs) to determine their abundance. Data for selected taxa will be confirmed by qPCR [40]. 
Alpha and beta diversity indexes will be calculated. The metagenome will be inferred from compositional data in 
silico. [41].  
Plasma Analyses. Plasma samples for glucose and insulin will be separated by centrifuge and the plasma 
immediately frozen at –80 C at the University of Toronto, in a locked room, in a locked building for analysis. The 
BBDC at the University of Toronto will perform analyses of the plasma glucose using the glucose oxidase method 
[42]. The Guelph Research and Development Centre will perform the analysis for the plasma insulin using 
enzyme-linked immunosorbent assay (ELISA), a solid phase two-site enzyme immunoassay method using 
Mercodia AB Insulin ELISA (Uppsala, Sweden) kits [43]Plasma glucose and insulin curves will be plotted as the 
incremental change over time and iAUC will be calculated geometrically for each participant using the trapezoid 
method, ignoring areas below the fasting value  [44].  J. Alick Little Lipid Research Laboratory at St. Michael’s 
Hospital will perform analyses of lipids (enzymatic method). ALT will also be analyzed at St Michael’s Hospital 
Core Lab. Remaining samples will be stored at -80 C for future analysis of metabolomics. The Matsuda ISI 
(Matsuda ISIOGTT) will be calculated using the 75g-OGTT derived plasma glucose (PG) and insulin (PI) valuess, 
according to the formula by Matsuda et al. [35]: 10 000 divided by the square root of ([FPGFPI]  ([mean PG  
mean PI]), where PG is expressed in mg/dl (0.0551mmol/L) and PI in U/ml (6pmol/L). The early insulin secretion 
index (∆PI30-0/∆PG30-0) will be calculated as the change in PI from 0 minutes to 30 minutes divided by the change 
in PG over the same period [45].  

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Biomarkers of adherence. Biomarkers of adherence will be assessed at the Department of Nutritional Sciences, 
University of Toronto by a novel gas chromatography, compound specific isotope ratio mass spectrometry (GC-
CSIRMS) method that detects 13C/12C ratios in serum fatty acids (e.g. palmitoleic acid) derived from the de 
novo lipogenesis (DNL) of added sugars from sugar cane (sucrose) or corn (high fructose corn syrup [HFCS]) will 
assess adherence to SSBs (increased) or water (decreased). A liquid chromatography with mass spectrometry 
using electrospray ionization (LC-ESI-MS/MS) will be used to assess urinary sucralose or acesulfame potassium 
(Ace K) [46]. Urinary fructose will be measured by gas chromatography mass spectrometry (GS-MS) [47].  
Questionnaires 
Dietary Assessment. Participants will be asked to fill out a weighed 3DDR using Nutritics, a nutrition software 
available for use on smartphones. The 3DDR will consist of two non-consecutive weekdays and one weekend 
day during the run-in phase and during the last week of each intervention. If participants do not have access to a 
smartphone, a paper form of the 3DDR will be provided. Participants will also be provided with scales to insure 
the accuracy of the record. Registered dietitians will instruct and review 3DDRs with participants. The 3DDRs will 
be used to evaluate changes in diet quality and will be analyzed by using Nutritics. Food cravings, hunger, and 
satiety will be assessed using modified Corby Martin’s Questionnaires [48].  
Behavior of beverage consumption will be assessed with a product consumption questionnaire. This will be 
done at the last visit for the participants that are still enrolled in the study and via telephone or email for the 
participants that have completed the study. 
 
 
Radiology 
Liver and calf muscle fat. We will use a 3T Siemens Magnetom Skyra scanner (Erlangen, Germany) with either an 
18 channel body phased array coil or a dedicated peripheral phased array coil. Single voxel, 30mm cubed 
spectroscopy will be acquired for IHCL and IMCL using a STEAM (Stimulated Echo Acquisition Mode) sequence 
and the following acquisition parameters: TR 3000 ms, multiple TE values 12 ms, 24 ms, 36 ms, 48 ms, 72 ms, 
mixing time (TM) 10 ms, 10 averages, 1024 data points and receiver bandwidth 1200 Hz for an acquisition time 
under one minute. Participants will be scanned for 30 minutes. 
Our Postdoctoral Fellow (Dr. Tauseef Khan, MBBS, PhD) will coordinate all analyses and our MSc candidate #2 
(Sabrina Ayoub-Charette ) will conduct fecal microbiome analyses. 
10.14 Study duration. Each participant will be enrolled for approximately 6 months (~164 days) and will undergo 
1 x 2-week run-in phase, 3 x 4 week interventions and 2 x 4 week wash-out phases. All participants will undergo 
each intervention for four weeks, the order being randomly assigned. The entire study is expected to take 
approximately 4 years to complete, allowing for ethics and material/data transfer agreement approvals (6 
months), rolling recruitment (6-months), run-in phase, intervention (x3) and washout (x2) phases (total 6-
months), microbiome/ plasma/ imaging/ biomarker analyses (9-months), data analyses (9-months), and final 
write-up (6-months). 
10.15 Data Integrity/Management. All paper copies of data forms will be stored under a double lock system to 
ensure confidentiality (locked cabinet in a locked office). Only PI, co-investigators and assigned study personnel 
will have access to the cabinet. The participant digital database will be securely stored centrally at SMH. The 
data management system enables anonymized and secure information storage, retrieval and sharing and will be 
organized and supported by the Li Ka Shing Knowledge Institute IT team of St. Michael’s Hospital. All data forms 
will be stored under a double lock system to ensure confidentiality. Only the PI, co-investigators and assigned 
trial personnel will have access to the cabinet. The Applied Health Research Centre (AHRC), St. Michael’s 
Hospital, will be contracted to manage the digital database securely and ensure data integrity. Their data 
management system enables anonymization, secure information storage, retrieval, and sharing of data.  
10.15 Personal Health Information. We will be collecting personal health information during this study. The 
personal health information will include name, phone number, email address, demographic information, health 

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history, questionnaires and records, and research visit records. In order to keep participant data confidential, we 
will create a master linking log which will separate identifying data (name and contact information) from study 
data with a study ID. The master linking log will be kept in a password-protected file on a drive through St 
Michael’s Hospital computer, only accessible to study staff and investigators. Any subject requesting access to 
their personal health information will be able to do so by requesting this through the study coordinator.  
 
10.16 Subject Compensation. Once participant is randomized travel compensation will be provided. To 
compensate for lengthy time required for study visits, each participant will be compensated $80 for first 5 visits 
and $95 for final visit, totaling $495 for completing the trial.  Participants will receive payment at the end of each 
phase (three times during the study). However, they will be informed that if they choose to withdraw early, they 
will be compensated for each visit that was completed. Transportation costs (i.e. parking or TTC tokens) will be 
reimbursed and breakfast will also be provided at each study visit. Any transportation costs incurred for 
additional visits for obtaining stool and urine collection kits will be reimbursed as well.  
10.17 Anticipated Challenges 
Recruitment challenges. We require individuals who are regular SSB drinkers. In fact, soft drinks are the second 
largest source of added sugars for adults in Canada [49] and the per capita intake of SSBs in Canada is 0.9 with 
estimated 20 percent drinking more than 1 SSB serving per day [50]. Even if half of this population drinks NSBs, 
there is estimated 10 percent adult population that drinks more than 1 SSB serving per day. In addition, we plan 
to recruit from overweight and obese individuals who are known to have a higher SSB intake [51].  
Compliance challenges. In dietary studies, compliance is usually a challenge. We will address this by having 
participants keep beverage logs at all phases of the trial including run-in and washout phases. Participants will 
be able to select a brand of beverage they like in both NSB and SSB arms. These beverages will be provided to 
the participants from the study site for the first week of each phase and shipped via Starhawk express for the 
three subsequent weeks. In addition, adherence to treatment will be reinforced with regular phone calls and/or 
emails to the participants. Since the intervention consists of a wide range of easily available supermarket drinks 
(Table 3), we do not anticipated challenges in acceptance on basis of palatability, taste or volume.  
Protocol compliance. Those who use antibiotics during an intervention phase will have to go through a 30 day 
washout period at the completion of their antibiotic course and restart that phase of intervention. If the 
antibiotic course is taken during the washout phase, a 30 day additional washout will be instituted. 
11. Statistical Analysis  
Data will be analyzed according to an intention to treat (ITT) principle using mixed models in STATA 14 
(StataCorp, Texas, USA). Sensitivity analysis will be performed on the basis of complete data availability for 
primary endpoints. A separate sensitivity analysis will be performed on the basis of antibiotic use during the 
trial.  
Primary outcomes. Repeated measures mixed effect models will be used to assess changes in the two primary 
outcomes i) beta diversity and ii) glucose iAUC between the groups. Pairwise comparisons between 
interventions will be performed using Tukey-Kramer adjustment or other appropriate statistics. For all primary 
outcomes effect modification by sex will be explored. We will use the truncated Benjamini-Hochberg false 
discovery rate controlling method with parallel gatekeeping procedure to correct for multiple comparisons for 
all primary outcomes [32].  
Secondary outcomes. Repeated measures mixed effect models be used to assess changes in weight, waist 
circumference, fasting glucose, 2hr plasma glucose, and MATSUDA. Pairwise comparisons between interventions 
will be performed using Tukey-Kramer adjustment or other appropriate statistics. For all secondary outcomes 

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effect modification by sex will be explored.  We will use the truncated Benjamini-Hochberg false discovery rate 
controlling method with parallel gatekeeping procedure to correct for multiple comparisons for all secondary 
outcome comparisons [33] if at least one primary outcome reaches significance. If none of the primary 
outcomes reach significance, the secondary outcomes will be analyzed as exploratory variables with no 
adjustment for false discovery rate.  
Exploratory and adherence outcomes. Repeated measures mixed effect models will be used to assess changes 
in all exploratory outcomes without controlling for false discovery rate. Pairwise comparisons between 
interventions will be performed using Tukey-Kramer adjustment or other appropriate statistics. Effect 
modification by sex will be explored. 
 
Subgroup Analysis. A priori analysis will be conducted by age, sex, ethnicity, baseline BMI, baseline WC, baseline 
FPG, baseline 2hPG (OGTT), baseline iAUC, medication use, sweetener blend (intervention), beverage type 
(intervention), and background sweetener use. 
12. Significance of work 
Replacement of SSBs is an essential recommendation in all dietary guidance on limiting added sugars. However, 
the role of NSBs to replace SSBs is complicated by its purported adverse effect on the composition of the gut 
microbiome with associated development of adverse glucose tolerance. Our study will be the first to investigate 
this question in humans in a randomized controlled trial comparing SSBs, NSBs and water. It will clarify if NSBs 
are similar to water and will inform the recommendations for dietary guidelines on replacements of added 
sugars such as SSBs. The proposed project will aid in knowledge translation related to the health effects of NSBs 
as alternative to sugars, informing evidence-based guidelines and improving health outcomes by educating 
healthcare providers and patients, stimulating industry innovation, and guiding future research design.  
 
13. TIMELINE 
Milestone 
Date 
Grant awarded 
Ethics Approval 
MTA/DTA Approval 
Recruitment commenced  
First patient randomized 
Recruitment completed 
Last patient completed 
Laboratory analysis completed 
Data analyses completed 
Manuscript completed 
Manuscript submitted   
Manuscript accepted 
Apr 2017 
Jan 2018 
Feb 2018 
Feb 2018 
Mar 2018 
Aug 2018 
Mar 2019 
Oct 2019 
July 2020 
Nov 2020 
Dec 2020 
Jan 2021 
 
14. BUDGET 
BUDGET ITEM 
SUBTOTAL 
PERSONNEL 
$150,000.00  
MICROBIOME 
$30,000.00  
PARTICIPANTS 
$42,000.00  
LABORATORY 
$70,000.00  

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BEVERAGES 
$3,000.00  
OFFICE SUPPLIES 
$2,500.00  
RECRUITMENT 
$20,000.00  
KNOWLEDGE TRANSLATION 
$12,500.00  
GRAND TOTAL 
 $330,000.00  
 
15. Genetic Testing: This aspect of the study will be optional and is not part of the main objectives of the 
study 
Objective: To determine whether there are any genetic differences between individuals in their response to 
study beverages. 
Background. There is considerable interest in the scientific community to study how genes may influence 
response to diet.  In terms of taste our diets tend to be bland containing lower salt and also less sugar.  The 
genes controlling these tastes may therefore determine intake [52, 53]. This is an example of why 
comprehensive genetic analysis, due to the implication of a growing number of genes of interest, would be 
helpful. We will, however limit our analyses to specific genes currently known to affect dietary response. 
Generally, the effect of genes on diet is very modest and usually requires significant numbers of people to detect 
the difference. It therefore has very limited clinical relevance. We may therefore pool data from across our 
future studies (typically 30-80 participants per study). A bio-bank will be established for the proper storage and 
management of these samples. 
We hope to make our initial findings known to participants at the end of the study, especially if we discover 
specific groups for which certain types of dietary advice may prove advantageous. Genetic testing results are 
solely for use in this study and will not be shared with any third parties (i.e. insurance companies or the 
hospital). 
Analyses. The DNA will be extracted from “buffy coat” of white blood cells obtained after plasma has been 
removed from citrated blood samples drawn at week 0 of phase I or any time point during the main study. 
Therefore, no additional blood samples will be required for this study.  
The genes of interest will include those related to the effectiveness of dietary change on blood glucose, 
cholesterol and associated measurements including blood pressure, taste preference, and other related genes 
which may influence response to diet.  
Incidental findings. An incidental finding is a finding concerning an individual research participant that has 
potential health or reproductive importance and is discovered in the course of conducting research but is beyond 
the aims of the study[54]. Any incidental findings (as defined above) obtained from screening or during the 
course of the main study from laboratory tests, ultrasound or MRI images will be provided to the family 
physician for further investigation. The participant will be notified in reassuring terms and advised to follow-up 
with their family physician. In this sub-study, if genes identified have clinical implications the advice of experts in 
that particular field will be sort to arrange appropriate counseling for the participant. The family physician’s 
involvement would be sought if the participant consents to it. However, the chances of incidental findings are 
limited since only specific genes known to affect dietary response will be studied. 
Banking of Samples. Blood samples (de-identified) will be stored at the University of Toronto (at the Medical 
Science Building or the Fitzgerald Building) until all planned analyses have been undertaken-usually about 3 to 5 
years. Optional longer-term banking will be made available to all participants for storage periods of 30 years 
after study completion to allow for use in future secondary and exploratory analyses in relation to the objectives 

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of the current research project. At the end of each storage period, samples will be destroyed by autoclaving. 
We are currently investigating the process of setting up a Biobank to efficiently and securely store and manage 
these samples; the necessary application will be sent to the St. Michael's REB once the project proposal is drawn 
up. Presently the use and ultimate destruction of these samples will be the responsibility of the investigator, Dr. 
Sievenpiper. However, detailed information on the organization and management will be in the biobank 
proposal that will be submitted to the REB in the near future. 
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