Effect of sugar-sweetened beverages onbaseline characteristics of the Double-blin Kids

MarLothar David Jan Kuijper, MartijnDepartment of Health Sciences, EMGO Institute for Heal1081 HV Amsterdam, the Netherlands

a r t i c l e i n f o

is 0.72, which corresponds with a difference in body weight of 2.3 kg.

Contemporary Clinical Trials 33 (2012) 247257

Contents lists available at SciVerse ScienceDirect

Contemporary C

j ourna l homepage: www.e lsevDiscussion: The double-blind design eliminates behavioral factors that affect body weight. Ifchildren gain less body fat when drinking sugar-free than when drinking sugar-sweetenedbeverages that would show that liquid sugar indeed bypasses biological satiation mechanisms.It would also suggest that a reduction in liquid sugars could decrease body fat more effectivelythan reduction of other calorie sources.

2011 Elsevier Inc. All rights reserved.

1. Introduction

Obesity in children has become a major health problem

Financial disclosure: Financial supportwas obtained fromThe NetherlandsOrganization for Health Research and Development (ZonMw) (grant#120520010; http://www.zonmw.nl/en/), the Netherlands Heart Foundation(grant #2008B096; http://www.hartstichting.nl/), and the Royal NetherlandsAcademy of Arts and Sciences (KNAW http://www.knaw.nl/smartsite.dws?

id=25792&lang=EaNG). The funders had no role in stution and analysis, decision to publish, or preparation ofdustry funding is involved.

Corresponding author at: VUUniversity, Faculty of EDe Boelelaan 1085, 1081 HV Amsterdam, the Nethe5983521; fax: +31 20 5983668.

E-mail addresses: J.C.de.Ruyter@vu.nl (J.C. de Ruyt(M.R. Olthof), L.D.J.Kuijper@vu.nl (L.D.J. Kuijper), kata(M.B. Katan).

1551-7144/$ see front matter 2011 Elsevier Inc. Adoi:10.1016/j.cct.2011.10.00718 months. We perform body measurements at 0, 6, 12 and 18 months. The primary outcomeis the z-score of BMI for age. The maximum predicted difference in this score between groupsKeywords:Randomized double-blind controlled trialSugar-sweetened beveragesChildrenObesityBMI for age z-scoregreet Renate Olthof,Bernard Katan

th and Care Research, VU University Amsterdam, Netherlands, VU University Amsterdam, De Boelelaan 1085,

a b s t r a c t

Background: Intake of sugar-sweetened beverages is associated with overweight in observa-tional studies. A possible explanation is that liquid sugars do not satiate and that their intakeis not compensated by reduced caloric intake from other foods. However, evidence from inter-vention studies for this hypothesis is inconclusive because previous studies were not blinded.Hence results may have been influenced by expectations and behavioral cues rather than byphysiological mechanisms.Methods: We designed the Double-blind, Randomized INtervention study in Kids (DRINK) toexamine the effect on body weight of covertly replacing sugar-sweetened by sugar-free bever-ages. Children were only eligible if they habitually drank sugar-sweetened beverages. Werecruited 642 healthy children (mean age 8.2, range 4.811.9). We designed, tested and pro-duced custom-made beverages containing 10% sugar and sugar-free beverages with thesame sweet taste and look. Children receive one 250 mL can of study beverage daily forArticle history:Received 17 June 2011Received in revised form 29 September 2011Accepted 18 October 2011Trial was deblinded 3 November 2011Available online 25 October 2011Janne Catharine de Ruyter,dy design, data collec-the manuscript. No in-

arth- and Life Sciences,rlands. Tel.: +31 20

er), M.R.Olthof@vu.nln99@falw.vu.nl

ll rights reserved.body weight in children: design andind, Randomized INtervention study

linical Trials

i e r .com/ locate /conc l in t r ia lworldwide. In the past three decades the prevalence of over-weight in children has increased dramatically [1]. Recentlythe prevalence of high body mass index in children appearedto plateau [2] but the number of overweight children remainshigh. Obesity in children is a risk factor for adult obesity, type2 diabetes, cancer, cardiovascular diseases and death before55 years of age [38]. Obesity in children also has negativehealth consequences during childhood itself such as insulin

248 J.C. de Ruyter et al. / Contemporary Clinical Trials 33 (2012) 247257resistance, hypertension, dyslipidemia and type 2 diabetes[9].

The increase in prevalence of obesity coincided with alarge increase in consumption of sugar-sweetened beveragein children [10]. Energy intake among children increasedsubstantially between 1977 and 2001 in the US, and energyfrom sugar-sweetened beverages accounted for more than50% of this increase [11]. Sugar-sweetened beverages, alsoknown as liquid sugars, are thought to be more fatteningthan sugars in solid form because they do not satiate [12].As a result children would not compensate for the intake ofliquid calories by eating less of other foods and drinks. There-fore sugar-sweetened beverages might increase total energyintake and cause overweight.

Indeed, in several observational studies intake of sugar-sweetened beverages was associated with weight gain both inadults [1315] and in children [1618]. However, some studiesfailed to find such an association [1921]. Also, an associationbetween sugar-sweetened beverages and weight gain may bedue to confounding because people who drinkmore soft drinksoften also eat more fast food and exercise less. Although moststudies adjust statistically for known confounders, residualand unmeasured confounding are of increasing concern in ob-servational nutrition studies [22,23]. Hence trials are neededfor conclusive evidence about causality.

Results of trials of the effect of liquid sugars on over-weight are inconclusive, with some finding an increase inweight [2427] while other trials found equivocal results[2830]. The lack of conclusive evidence may be due to alack of proper placebo treatments, small samples sizes,short duration and lack of individual randomization. Also,none of these trials was properly blinded. Blinding is crucialbecause without it, results may be biased by effects on foodintake of behavioral cues and expectations rather than byphysiological mechanisms [31].

Hence there is a need for conclusive evidence whetherliquid sugars fail to evoke compensatory changes in food intakeand are therefore more fattening than other sources of calories[32,33]. We designed DRINK the Double-blind, RandomizedINtervention study in Kids to examine the effect of covertlyreplacing sugar-sweetened beverages by sugar-free beverageson bodyweight. This allows us to study biological compensato-ry mechanisms independent of behavioral cues and voluntarychanges in intake. In this article we describe the rationale,design, methods and baseline characteristics of this study.

2. Methods

2.1. Outline

DRINK is an individually randomized, double-blind, con-trolled, parallel intervention study in free-living school chil-dren. For 18 months, 642 school children randomly receivedaily either one can of sugar-free beverage (treatment) orone can of sugar-sweetened beverage (control). We wouldhave preferred to use water as the treatment beverage, butthat would make blinding impossible. Children consume thebeverage at school on weekdays during their morning break,and at home during weekends and holidays. The study isdouble-blind; neither the researchers nor the children, parentsor teachers know who drinks which beverage. The study isconducted in the Netherlands at eight schools in an urbanizedarea near Amsterdam. We distribute the beverages to theschools (bi)weekly by courier. The study was preceded by apilot study to test feasibility and logistics and by a sensorystudy (see Formulations) to help us in developing the beverages.

2.2. DRINK pilot study

The pilot was a two-month study conducted in AprilMay2009 at one elementary school in Purmerend, 20 km fromAmsterdam. The study was conducted to the guidelines laiddown in the Declaration of Helsinki and the study protocolwas approved by the Medical Ethical Committee of VU Uni-versity Medical Centre Amsterdam. Written informed con-sent was obtained from a parent or guardian. In the rest ofthis paper parent will refer to both parents and guardians.The inclusion and exclusion criteria for the pilot were thesame as for the main study (see Recruitment). Forty-fourchildren aged 512 were randomized to receive daily one330 mL can with either sugar-sweetened or sugar-free bever-age (bought from Unilever, Colworth, UK). Both were avail-able with lemon and peach flavor. Our courier transportedthe beverages to the school.

We asked various stakeholders for advice on how to set upthe main DRINK study. Frequent conversations with the chil-dren taught us that offering more than two flavors wouldhelp to increase compliance, and that newsletters, contestsand birthday cards would be appreciated. Therefore weimplemented these and other forms of entertainment in themain study. The pilot lacked entertainment except the possi-bility to collect fancy stickers that we attached to the cans inthe last two weeks. In an evaluation form teachers reportedthat it was not a problem for them to accommodate consump-tion of our beverages at school. The parents reported that thechildren liked the pilot and that they were keen on bringinghome the weekend and holiday cans. Parents also broughtup that it would be impossible to transport cans during thesummer holidays of 6 weeks. Therefore we interrupted treat-ment in the main DRINK study for 6 weeks in the summer of2010, and prolonged it by 6 weeks at the end. Parents alsodid not like to return empty cans used at home back to schoolduring the pilot. Hence we collect cans used at school but notcans used at home to assess compliance during the mainstudy (see Ancillary measurements). Our advisory boardsuggested that schools might expect something in return fortheir participation. Therefore we offered them health relatededucation materials. Two schools made use of this option.

2.3. Design and setting of main DRINK study

The study protocol was approved by the Medical EthicalCommittee mentioned above. DRINK is an individually ran-domized, double-blind, controlled, parallel interventionstudy in free-living school children aged 511 years (Fig. 1).All children receive 250 mL of the study beverage per daywhich provides either 0 g or 25 g of sugar. Children in thecontrol group do not change their liquid caloric intake be-cause they replace their customary sugar-sweetened bever-age brought from home by a sugar-sweetened studybeverage. Children in the treatment group replace their cus-tomary sugar-sweetened beverage by a sugar-free study

ention

249J.C. de Ruyter et al. / Contemporary Clinical Trials 33 (2012) 247257beverage. Therefore they consume 100 liquid calories per dayless than the control group.

We conduct the study at eight elementary schools, in theZaanstreek, Purmerend and Haarlem, in an urbanized area,1633 km from Amsterdam. It is customary for children inDutch elementary schools to consume a beverage broughtfrom home in class during a morning break around 10 am

Fig. 1. Flow of recruitment and design of the Double-blind Randomized INtervon body weight.under supervision of the teacher. We replace the beveragebrought from home by a study beverage and ask the teacherto check if children consume their beverage during the morn-ing break and to remind them to take home weekend andholiday cans. Parents supervise consumption of the studybeverage on weekend days and during holidays.

2.4. Beverages

2.4.1. FormulationsThe development and manufacture of the beverages was

the major challenge for our study. We needed pairs of bever-ages, one sugar-free and one sugar-sweetened, which tastedand looked the same. The beverages needed to be safe andstable, attractive and pleasant-tasting for children. They alsohad to be acceptable for parents and schools. We opted fora non-carbonated beverage because of the poor healthimage of fizzy drinks. We offered several flavors raspberry,lemon, peach and mango to increase compliance.

Before the study started we tested the sensory qualities ofthese beverages in 89 children aged 512 in Purmerend atthe same school where we did the pilot study. The childrenliked the sugar-free beverages and sugar-sweetened bever-ages equally.

The sugar-sweetened beverages contain 25 g of sugar and100 kcal per 250-mL can (Table 1). The sugar-free beveragesdo not provide calories. They contain the artificial sweetenerssucralose (0.157 g/L) and Acesulfame-K (0.057 g/L). Thesesweeteners are approved by the US Food and Drug Adminis-tration, the Joint Commission of Experts on Food Additives ofthe World Health Organization, the Food and AgricultureOrganization, and the European Food Safety Authority. Theacceptable daily intake (ADI) for sucralose is 15 mg/kg body-weight/day [34]. This means that a child of 20 kg can drink

study in Kids (DRINK) that studies the effect of sugar-sweetened beveragesseven cans of sugar-free study beverage per day. The ADIfor acesulfame-K is 9 mg/kg bodyweight/day [35] which cor-responds with 13 cans. Since we offer one can per day, theamount of artificial sweeteners is safe.

We use vegetable and fruit concentrates as colorings inthe mango, peach and raspberry flavored beverages. Thelemon drinks do not contain colorings.

2.4.2. ProductionWe hired JJM (Brussels, Belgium) to design the cans,

Crown (Wantage, United Kingdom) to make the cans, and

Table 1Composition of study beverages in the Double-blind, Randomized INterven-tion study in Kids (main DRINK study) a,b.

Sweetener Calories

Sugar-sweetened beverageper 250-mL can

Sugar 25 g 100

Sugar-free beverageper 250-mL can

Sucralose 0.04 g 0Acesulfame-K 0.01 g

a Ingredients besides the sweetener: fruit flavor (lemon, mango, raspber-ry or peach), fruit juices (lemon, mango, raspberry or peach), colorings (GNTExberry vegetable and fruit concentrates; no color in lemon), water, triso-dium citrate, citric acid, ascorbic acid, malic acid (malic acid only in sugar-sweetened beverage).b We bought the colorings from GNT (Mierlo, The Netherlands) and fla-

vorings from Unilever (Colworth, UK). All other ingredients were fromRefresco Benelux (Maarheeze, Netherlands).

Refresco Benelux (Maarheeze, Netherlands) to produce thebeverages and fill the cans. Fig. 2 presents the can design.We used 250 mL cans because that fitted better with the fac-tory's logistics than 330 mL cans and because the shelf life ofcans is better than that of cardboard containers. Each of thefour flavors was produced as a sugar-sweetened and asugar-free variant. The first batch consisted of 30000 cansper flavor/sweetener combination for a total of 240000 cans,plus 20000 cans of sugar-sweetened peach beverage for therun in period. These were produced on 24 August 2009,prior to the start of the study. The second batch of 200000cans, i.e. 25000 per flavor/sweetener combination, was pro-duced on 7 September 2010. The beverages will never beavailable commercially. VU University has registered thebrand name (Blikkie, colloquial for little can) and the candesign to prevent commercial exploitation by third parties.

2.5. Sample size calculation

We converted the predicted difference of change inweight into a BMI for age z-score because in children the re-lation between BMI and body fatness depends on age andgender, while the BMI for age z-score does not. Hence theBMI for age z-score or standardized BMI allows comparisonof children of different ages and gender.

We estimated the difference of change in BMI for age z-scorefrom the expected difference in weight change. At the end ofthe study our subjects will on average be aged 9.5 years. Themean weight of Dutch children aged 9.5 years is 32.25 kgtheir mean height is 1.408 m and mean BMI 16.24 kg/m2 [40]A weight reduction of 2.3 kg will produce a weight of 29.95 kgand a BMI of 15.11 kg/m2. Thus the maximum expected effectof the treatment is a reduction of 1.13 kg/m2. In Dutch childrenof this age, a fall of 1.13 kg/m2 in BMI will shift a child down-wards in the distribution by 0.72 standard deviations [40].

The effect of removing sugary drinks on health could still berelevant if only part of the sugar removed was compensated byincreased intake of calories from other sources. Also, we cannot

is colloht side

250 J.C. de Ruyter et al. / Contemporary Clinical Trials 33 (2012) 247257The sample size calculation was based on the expected ef-fect of treatment on the change of the z-score BMI for age andits SD. BMI stands for BodyMass Index and is the weight divid-ed by height squared (kg/m2).The z-score is the number ofstandard deviations by which a child differs from the meanBMI of children of the same age and gender. We calculatedthe number of children required as N=7.92(within-person SD of the change in z-score with time/expected differ-ence of change in z-score) (power=0.8; =0.05) [36].

We calculated the expected difference of the change inweight and z-score between treatments from the differenceof sugar intake. Children in the treatment group reduce theirintake of liquid sugar by 25 g per day which equals 12 600 gof sugar or 50400 kcal in 18 months. A naive calculationwould suggest that at 7709 kcal per kg weight change [37]this would equate a difference of 6.54 kg. However, any reduc-tion in weight reduces the basal metabolic rate and the energycosts of activity [38]. Thereforewe predict that themean differ-ence of change in weight between the treatment and controlgroup is 2.3 kg, if children receiving sugar-free drinks do notcompensate for the absence of sugar by eating and drinkingmore of other foods [39].

Fig. 2. Can design of the sugar-free and sugar-sweetened beverages. Blikkiesearch team and logo of the study and VU University are depicted on the rigexpect 100% compliance to treatment.Wedesigned the study sothat we would still pick up a difference if children in the sugar-free group compensated 50% of the calories lost from sugar byincreased intake of other calorie sources, and if all participantsonly consumed 50% of the drinks. The predicted difference ofchange in z-score then becomes 50%50%0.72=0.18.

We calculated the within-person SD of the change in z-score from the changes in height and weight of 1017 partici-pants in the ChecKid study [41]. ChecKid examines trends inoverweight and obesity among children in Zwolle initiallyaged 412 years. Using raw data obtained from childrenwhen they were aged 6.7 and 9.7 we calculated that thechange in BMI for age z-score over three years, relative tothe Dutch mean averaged 0.03 and its SD equaled 0.66.

Hence our sample size calculation is: N=7.92(0.66/0.18)=212 subjects per treatment. We recruited 642 be-cause we anticipated a dropout of one third.

2.6. Recruitment

We recruited participants between August and November2009 (Fig. 1). We first approached the boards of seven school

quial Dutch for little can. Telephone number and email address of the re-of the can. Expiration date is imprinted at the bottom of each can.,.

2.7. Baseline characteristics

Most of our 642 participants have a normal weight(Table 2). Their mean BMI for age z-score is 0.03 where 0 isthe Dutch average in 2009. The number of non-western chil-dren in our population is slightly higher than that in theDutch population as a whole [44]. In 44% of the householdsthe highest education level attained by either parent isIntermediate vocational education or less, and 55% had aHighschool or college degree. The level of education is slight-ly higher than the Dutch average [45].

2.8. Randomization of participants and beverages

Randomization was done by a statistician (L.D.J.K.) who isnot involved in the execution of the study. Eligible childrenwere individually randomized. An Excel visual basic macro

Table 2Baseline characteristics of the 642 children in the Double-blind RandomizedINtervention study in Kids (DRINK).

Characteristics n (%) or MeanSD

No. of children 642Girls 301 (46.9%)Age (years) 8.21.9Ethnicitya

Dutch 502 (78.2%)Non-western 120 (18.7%)Other 12 (1.9%)

Household highest educational level(Dutch translation)b

Elementary (primary) school(Lagere school/basisonderwijs) 6 (0.9%)Lower vocational secondary educationor technical secondary education(LBO, LTS, LEAO, Lagere tuinbouw)

30 (4.7%)

Intermediate secondary education(MAVO/MULO)

51 (7.9%)

Intermediate vocational education(MBO, MBA, LO-akten, MTS, MEAO)

195 (30.3%)

Highschool (HAVO/VWO, MMS, HBS) 92 (14.3%)Higher vocational education/collegedegree (HBO/Universiteit)

259 (40.5%)

Weight (kg) 30.198.86Height (cm) 132.512.6BMI 16.82.6BMI for age z-score 0.031.04

251J.C. de Ruyter et al. / Contemporary Clinical Trials 33 (2012) 247257districts that encompassed 131 schools for permission tocontact their schools. We approached another 11 schools di-rectly. Out of these 142 schools, 22 were willing and able tocooperate and granted us access to parents and children.The 4913 pupils at these 22 schools received a recruitmentflyer including a consent form. We set up information boothsat the schools to tell parents and children about the study.We also let them taste the study beverages and we sent outa press release which led to broad exposure in the media.An independent physician was available for parents to an-swer health related queries. Parents of 1435 children (29%)gave written informed consent for their child to participate.We asked children to give verbal assent to the parent. Parentsthat gave their consent were asked to complete a question-naire so we could determine if the child was eligible. We con-sidered it unethical to increase the children's sugar intake.Therefore an important inclusion criterion was that childrenhad to habitually consume sugar-sweetened beverages dur-ing the morning break at school on at least three out of fiveschool days. Other criteria were:

Inclusion criteria:

Habitual consumption of 250 mL or more per day of sugar-sweetened beverages on at least three out of five schooldays

Minimum age of 5 years at the start of DRINK Young enough to be still in elementary school at the end ofDRINK

Written informed consent by a parent

Exclusion criteria:

Medication or medical treatment for obesity Diabetes, growth disorders, celiac disease, or serious gastro-enterologic diseases, e.g. inflammatory bowel disease

Medical history or surgery known to interfere with thestudy

Participation in another intervention study up to 3 monthsbefore and during DRINK if that interfered with our study

Physical disabilities that interfere with the measurements Plans to relocate and change school during DRINK

Because 1435 children volunteered and we only needed640, we excluded schools that were difficult to reach or hadfew prospective participants. This left 699 participants. Appli-cation of the inclusion and exclusion criteria left 642 childrento be enrolled and randomized (Fig. 1).

The questionnaire also contained demographic and ethnicquestions about the child and its parents. A child is consideredDutch if both parents are born in the Netherlands, Non-westernif one or both parents are born in Suriname, Dutch Antilles,Turkey or Morocco, or Other if both parents are born in a coun-try other than these. In case of a single parent household, weused the country of birth of this parent. We determined educa-tional level based on both parents, whichever was highest.Level of education was graded as: Elementary school, Lowervocational secondary education or technical secondary educa-tion, Intermediate secondary education, Intermediate voca-tional education, High-school graduate and Higher vocationaleducation/college degree. We used international cut-offs foroverweight and obesity in children and for low and healthyBMI [42] [43].Weight statuse

Low BMI 5 (0.8%)Healthy BMI 514 (80.0%)Overweight 101 (15.7%)Obese 22 (3.4%)

Skinfold thicknessc

Biceps (mm) 6.83.2Triceps (mm) 11.85.0Subscapular (mm) 8.24.8Supra-iliac (mm) 9.25.8Sum of skinfolds (mm) 36.017.8

Waist-to-height ratio (%) 44.44.0Electrical-impedance fat massd

(kg) 5.743.75(%) 17.836.87

a n=634; eight households refused.b n=633; nine households refused.c n=641; one child refused.d n=638; four children refused.e We used international cut-offs for overweight and obesity in children

and for low and healthy BMI [42] [43].

252 J.C. de Ruyter et al. / Contemporary Clinical Trials 33 (2012) 247257program randomly assigned children to sugar-sweetened orsugar-free beverages within each school so that mean age,gender and initial BMI were equal between treatments. Ran-domization was done with minimal human intervention, asfollows. Data of children who met all inclusion and exclusioncriteria were closed out in the central database (MSAccess) and exported per school into an MS Excel workbook.The Excel macro written by L.D.J.K. assigned all children arandomization ID number. Children in the same householdreceived the same randomization ID number and thus thesame treatment, because there is a risk that they drink eachother's study beverages at home. Children, parents andteachers have not been informed that siblings receive thesame treatment.

The macro then assigned to each randomization ID numbera random number sampled from a continuous uniform distri-bution between 0 and 1, e.g. 0.64451. The macro sorted chil-dren by this random number, and the upper half wasassigned to one treatment and the lower half to the other treat-ment. The program then calculated the following percentagesper treatment group: males; birth year 19981999; birth year20002001; birth year 20022004; BMIb15; BMI 1518 andBMI>18. Ideally all these percentages should come out 50/50. If any of these percentages was b45% or >55% the macroautomatically assigned new random numbers and reiteratedthe process until gender, birth year and initial BMI were evenlydivided between the treatments, i.e. within each school eachtreatment group contained more than 45% and less than 55%of each gender, birth year category and initial BMI category.Themacro ran from start to finishwithout human intervention.It reported the final randomization assignments, the percentdistributions of gender, birth year and BMI over treatments,and the number of iterations needed to achieve this. The statis-tician assigned the upper and lower half of the randomnumberdistribution to intervention or control treatment by a one-timeflip of a coin for the first school randomized; this assignmentthen held for all schools. When a school had an odd numberof participating households the assignment of the medianchild or household alternated from one school to the next.This procedure is equivalent to blocked randomization [46],but its automation is more straightforward and transparent.The statistician sent the treatment codes to the database man-ager who entered them into the central administration data-base. The database manager is not further involved in thestudy.

L.D.J.K also produced anonymous codes for the beverages.He randomly assigned the codes 1001 to 1008 to the eighttypes of beverages (4 sugar-sweetened, 4 sugar-free) andsent these to the factory. Beverages for the run-in period (allsugar sweetened) received code 1009. The factory printed thebeverages codes on the cardboard trays on which cans werepacked. The bottom of each can was imprinted with a 17-digit code into which the beverage code was encrypted. Thisencryption produced 100 different can codes per beveragetype, for a total of 800 can codes.

Personnel at the warehouse where cans are weeklyrepackaged for the children are inevitably aware which 4codes together form one treatment group, but do not knowwhat the treatment is. When cans are repackaged they areseparated from their cardboard trays, and the codes on thebottom of the cans are almost always different betweencans even if treatments are the same. Only our statisticiancan track down the treatment code for an individual can. Sofor parents, teachers, children and study researchers thecodes on individual cans cannot reveal the treatment.

Treatment assignments and beverage codes are held byL.D.J.K. and by the outside database manager. The University'sattorney holds a print of treatment assignments and beveragecodes in a sealed envelope.

2.9. Packaging and distribution of study cans

The cans were shipped from the factory to the warehouseof our courier in Egmond aan den Hoef, 50 km northwest ofAmsterdam, and stored at ambient temperature. BetweenApril and July 2011 the beverages will be stored at 18 C toprevent deterioration of taste.

At the warehouse, one researcher coordinates the repacka-ging of the cans according to a standardized procedure. Wepackage the cans per week per child into 112115.5 cmcardboard kid boxes imprinted with the study logo. A kidbox holds 8 cans: 5 for school days, 2 for weekend days and 1spare can. Each kid box also contains 8 name labels that chil-dren stick onto their cans each Monday morning to avoidmix-ups in class. The kid boxes are delivered to the schools in354416 cm cardboard class boxes, which hold up to 6kid boxes. Our courier distributes the boxes bi weekly orevery week if the school lacks storage space and brings backempty and unused cans. We distributed extra spare cans toall children at the beginning of the study to be used in case chil-dren forget to bring home their weekend and/or holiday cans.After the second production in September 2010 we distributednew spare cans.

2.10. Body measurements

Measurements are done according to a standardized pro-tocol at t=0, 6, 12 and 18 months. Children are measured intheir underwear during school hours, generally between8.30 am and 3.30 pm. We ask children to visit the bathroombefore the measurements. Children who stopped consumingthe study beverages continue to be measured if the parentand the child permit us to do so.

Two female researchers are always present simultaneouslyat eachmeasurement as chaperones.Wemeasure bodyweightto the nearest 0.1 kg on a Marsden MPMS-250 digital scale(Oxfordshire, United Kingdom). We calibrate the scale eachmeasurement day with a KERN 366-98 standard weight of20 kg (Balingen, Germany). We measure height to the nearestmillimeter with a SECA 214 (Hamburg, Germany). Height willbe measured in duplicate at 18 months. We measure waist cir-cumference twice to the nearest 0.1 cm at the midpoint be-tween the bottom rib and the top of the hipbone with a SECA201 flexible steel tape measure (Hamburg, Germany) [47].We measure biceps, triceps, subscapular, and supra-iliac skin-folds in triplicate with a Harpender Skinfold Caliper HSK-BI(Burgess Hill, United Kingdom) [47]. Two specially trained re-searchers perform the waist circumference and skinfold mea-surements. Each child is measured by the same researcherthroughout the study to reduce variation. We measure arm-to-leg electrical impedance twice with a BodyStat 1500MDD(Douglas, United Kingdom). We put 3M 2330 electrodes (St.

253J.C. de Ruyter et al. / Contemporary Clinical Trials 33 (2012) 247257Paul, U.S.A.) on the right hand and foot according to the manu-facturer's manual. Then we connect the two cable leads of theBodyStat to the electrodes and perform the measurementsaccording to standardized procedures [48]. We calibrate theBodyStat each measurement day with the BodyStat calibrator.The calibrator is a small device onto which we connect to thecable leads to perform ameasurement. The calibration has suc-ceeded if the impedance is between 496 and 503 .

2.11. Ancillary measurements

We measure compliance by counting returned cans andby analysis of sucralose in urine. We count cans from schooldays only, because parents do not return cans used at homeduring weekends and holidays. On school days childrenplace their empty cans back into their kid boxes, and theseare collected by our courier together with the unused cans.We check returned cans one week each month i.e. 25% of allcans returned. Cans are scored as empty, half-filled or full.

We will measure sucralose in urine of the children in thesugar-free group. A study in humans showed that 14.5% ofingested sucralose is excreted in urine [49]. Therefore wecan use sucralose in urine as a compliance marker. We willalso measure urine samples from children in the sugar-sweetened group as a control. Compliance is assumed to besimilar in both groups since the sugar-sweetened and thesugar-free beverages were equally appreciated by the chil-dren (see Formulations). Therefore we collect spot urinesamples from all children at t=0, 6, 12 and 18 months.

We administer a short dental questionnaire and a hedonicquestionnaire at t=12 and t=18 months. The dental ques-tionnaire inquires about the number of new dental fillings andteeth newly extracted because of caries. The hedonic question-naire asks: 1. Howmuch do you want to drink the study drink?2. How satiated do you feel? 3. What do you eat together withthe study drink? 4. How much do you like the study drink?

2.12. Endpoints

Our null hypothesis is that children in the sugar-free groupwill fully compensate for the loss of the sugar from their habit-ual drinks by increasing their intake of calories from otherfoods and beverages. Under this hypothesis wewill find no dif-ference between the sugar-free and sugar-sweetened group inthe change in BMI for age z-score between t=0 andt=18months. The alternative hypothesis is that compensa-tion is not 100% and that children receiving sugar-free bever-age will gain less body fat than those in the control group.

The primary endpoint is the difference between thesugar-free and sugar-sweetened group in the change in BMIfor age z-score between 0 and 18 months. The secondary out-comes are the waist-to-height ratio (%), the sum of the fourskinfolds (mm) and body fat percentage estimated from elec-trical impedance (kg and %). Waist-to-height ratio is an accu-rate measure of body fat in children that does not require sexand age specific adjustments [50]. Skinfold thickness is alsoan accurate indicator of body fat in children [51]. We willestimate fat percentage from the electrical impedance asfollows [52]: Body fat (kg)=body weight fat free mass.Fat free mass (kg)=0.622height2/R50 (Ohm)+0.234weight (kg)+1.166R50=resistance at 50 kHz.2.13. Data analysis and statistics

We consider a per-protocol analysis most suitable be-cause our research question examines the biological effectof sugar-free beverages on weight instead of the effectivenessto implement an intervention of sugar-free beverages. Wewill also do an intention-to-treat analysis to take into accountselective dropout and to prevent ignoring children whostopped due to effects of the study beverages on body weight.

Our primary analysis will thus be per-protocol analyses ofthose children who consumed the study beverages until theend of the study. The primary endpoint is BMI for age z-score, and the secondary endpoints are sum of four skinfolds(mm), waist to height ratio (%) and fat mass determinedfrom electrical impedance (kg and %). The difference inchange from 0 to 18 months between the sugar-free andsugar-sweetened group will be analyzed with the indepen-dent samples t-test. For each endpoint we will report themean difference in change between the sugar-free andsugar group and its 95% confidence interval and p-value. Tocheck the robustness of this analysis we will also perform alinear regression analysis that corrects for the outcome vari-ables at baseline. We do not expect that this will lead tomarkedly different results because we randomized the par-ticipants into two large groups and stratified for school, gen-der, age and initial BMI.

Approximately a third of the participants are siblings. Wewill perform subgroup-analyses in which we will collapsesiblings from the same household into one fictional subjectwho will be assigned the mean value of these siblings. Thiswill reduce the number of subjects and eliminate effects ofinterdependence between siblings.

We will also perform subgroup-analyses for the 80% mostcompliant children, where we exclude the 20% of the subjectswith the lowest empty can counts.

Secondary analyses will be intention-to-treat analyses.These analyses will include children who stopped drinkingthe lemonades prematurely but were available for the finalmeasurements at 18 months. We expect that final measure-ments will be available for 613 out of 642 children random-ized. We will not use the last observation carried forwardmethod or imputation techniques because there are manyobjections against these methods, especially in growing chil-dren. We will perform the same analyses on this dataset asdescribed above for the primary analyses.

The second and third timepoints, t=6 and t=12 months,will be used for graphs but not for analyses. The level of signif-icance will be Pb0.05 (2-tailed). Treatment codes will be bro-ken after blinded data-analysis. For the blinded data-analysiswe will use the numbers 0 and 1 for the treatment groups.After the analyses we will ask our statistician (LDJK) to de-blind the dataset.Wewill use the Statistical Package for the So-cial Sciences (SPSS) version 17.0 to perform the analyses.

2.14. Timelines

Fig. 3 presents the planning of the study. After the recruit-ment and baseline measurements we randomized the childreninto the two treatment groups. There were no dropoutsbetween the baseline measurements and randomization. Chil-dren started the study with a run-in period of one week in

Kids (

254 J.C. de Ruyter et al. / Contemporary Clinical Trials 33 (2012) 247257which they were all given the sugar-sweetened peach lemon-ade. This run-in period served to iron out remaining logisticproblems and did not contribute to the length of the interven-tion period. The first two schools started the intervention onNovember 14, 2009, another three schools on November 30,2009 and the final three schools on December 7, 2009. Thelast three schools started without a run-in period because of

Fig. 3. Study planning of the Double-blind Randomized INtervention study in6, 12 and 18 months, dental health questionnaires at t=12 and 18 months.time restrictions. We interrupted treatment during the sum-mer holidays of 6 weeks from July 10, 2010August 22, 2010.Final measurements are done after 18 months interventionand the study therefore ended in July, 2011.

3. Discussion

Low-caloric drinks seem a healthy alternative to sugar-sweetened beverages to prevent obesity in children becausesugar-sweetened drinks may fail to satiate and thereforeadd calories on top of the rest of the diet. However, conclu-sive evidence that sugar-sweetened beverages are moreprone than other foods to cause overweight is lacking. There-fore we designed the DRINK study. Our study examines ifchildren who are switched to sugar-free beverages increasetheir caloric intake from other sources to compensate thecaloric deficit in these beverages, or not.

3.1. Strengths and limitations

Our study has several strengths. First, the study is double-blind; nobody knows if a child is drinking sugar-sweetenedor sugar-free beverages. Therefore children in the sugar-freegroup receive no explicit or subconscious cues that they areexpected to lose weight, and children in the sugary groupare not encouraged to eat less or exercise more so as toavoid weight gain. Hence this study will be a strict test ofeffects of liquid sugars on body weight through unconsciousphysiological mechanisms that regulate food intake. Previoustrials in children [2830] and in adults [27] on the relationbetween sugar-sweetened beverages and body weight inves-tigated the effects of educational programs or environmentalinterventions. These trials were not blinded and could notseparate changes in body weight due to behavioral changes

DRINK). Body measurements and sensory evaluations are performed at t=0,from changes due to physiological factors. Other trials inadults [2426] on the relation between sugar-sweetenedbeverages and body weight were also not properly blinded.

A second strength is that treatment lasts 18 months andthat we have 642 participants. Other trials [2426] were rel-atively short, varying from 3 to 10 weeks, and sample sizesranged from 15 to 42 subjects.

A third strength is the use of a proper placebo treatment.A study on the effect of sugar-sweetened beverages onweight gain requires sugar-sweetened and sugar-free drinksthat taste and look the same. We managed to develop andproduce these beverages. Earlier trials used other treatments.Tordoff [25] used high-fructose corn syrup versus aspartamesweetened beverages, Raben [26] used sucrose-sweeteneddrinks and foods versus artificially-sweetened drinks andfoods, DiMeglio [24] used jelly beans versus caffeine-freesoda.

The fourth strength is the individual randomization,which will average out other behaviors that affect weight.For example, both groups should have similar numbers ofchildren that watch a lot of television or change their TVwatching habits, that eat a lot of candy, or that becomemore or less active. Also, parental education and ethnicityare similar for both experimental conditions. Furthermore,we stratified participants by school, gender, age and BMI cat-egory to ensure that these potential determinants of weightgain were evenly distributed between the treatment and

drates and weight status might consider measurements of

255J.C. de Ruyter et al. / Contemporary Clinical Trials 33 (2012) 247257caloric intake.Second, the ideal endpoint would have been fat mass esti-

mated from dual-energy X-ray scans. This was not feasible.However, if the study beverages cause differences in BMIfor age z-score it is plausible that these are due to differencesin fat mass, and we have other body measurements to backthis up.

Third, our primary compliance marker is counts ofreturned empty cans that children consumed during schooldays. We did not collect empty cans from the weekendsand holidays. In the sugar-free group analysis of sucralosein urine will provide a measure of compliance that is notavailable for the children receiving sugar-sweetened lemon-ade. However, our objective measurements of compliancestill exceed those in most other trials of diet and body weight.

Fourth, we excluded schools that were difficult to reach orhad few prospective participants. We do not think that thishas an effect on the generalizability of the results. Werecruited schools from an urbanized area near Amsterdam.We included the children from six towns and excluded chil-dren from six towns that partly overlapped. Based on zipcodes of schools included and excluded, socioeconomic sta-tus is quite similar and close to the average Dutch socioeco-nomic status. In addition we do not study behavior but aphysiological mechanism that in principle should not bemuch sensitive to socioeconomic status or place of residence.

The study population consists of healthy children and fo-cuses on biological satiation mechanisms that may be widelyshared. We therefore assume that findings of this study canbe extrapolated beyond the healthy Dutch children of ourstudy. On the other hand the effect of sugar-sweeteneddrinks on body weight might depend on body size, genetics,ethnic factors or health, and thus our findings should be ex-trapolated with caution and confirmed in other populations.

3.2. Interpretation of outcomes

There are two possible outcomes of our study. If we find nodifference in weight gain between the sugar-free and sugar-sweetened group, then children in the sugar-free group musthave increased their caloric intake from other sources tocompensate for the calories lacking from the study beverage.An exclusive focus on sugar-sweetened beverages might thenbe less effective to decrease overweight in children. Other in-terventions than reducing intake of sugar-sweetened bever-ages to decrease overweight in children should receive equalemphasis. Still, a lower intake of sugar-sweetened beveragescontrol group. Therefore we assume that any difference inchange in endpoints can be attributed to the study beveragesand not to other life style factors.

Our study also has limitations. First, the hypothesis thatwe test deals with satiation and food intake, and ideally weshould measure what the children eat and drink in responseto the treatments. However, it is impossible to measure foodintake in free living subjects to the degree of precision re-quired for our study. As body fatness is the outcome of con-cern, we decided to concentrate on this endpoint. Follow-upexperiments with smaller groups that examine the underly-ing mechanisms of the association between liquid carbohy-would help to prevent weight gain because they are a majorsource of calories.

If both treatments have the same effect on weight it couldalso be argued that the artificial sweeteners in the sugar-freebeverages stimulated caloric intake [53]. Some animal studiessupported this hypothesis [5456] but experiments in humans[5759] did not. The hypothesis that artificial sweeteners in-crease energy intakewould therefore not be themost likely ex-planation if we find no difference between the groups.

If we find that weight gain slows after removal of liquidsugar then children in the sugar-free group evidently didnot increase their intake of other foods and drinks sufficientlyto compensate their reduced intake of liquid calories. Itwould also suggest that the converse holds true, i.e. that add-ing liquid sugar to the diet causes weight gain because it isnot spontaneously compensated by reduced intake of otherfoods. That would corroborate the liquid sugar theory andsuggest a unique role for liquid sugars in causing weightgain in children. This outcome would further support currentrecommendation to discourage sugar-sweetened drinks andpromote non-caloric drinks such as water to prevent weightgain in children. However, one piece of evidence would stillbe missing: does covert removal of calories from solid foodsalso lead to weight reduction? Current theory suggests thatit would not, and that such calories would be compensatedfor from other foods. However, only a double blind trial sim-ilar to ours can provide decisive evidence on this reduction.

Conict of interests

The authors declare that they have no conflict of interest.

Author contributions

J.C.R. coordinated the pilot study, sensory study and themain trial. She also supervised the data collection, co-obtained medical ethical approval, supervised data manage-ment, was responsible for the recruitment of subjects, man-aged logistics of the trial and drafted the manuscript. M.R.O.co-obtained funding, obtained medical ethical approval, con-tributed to the design, co-supervised the study and co-supervised the development and manufacture of the bever-ages. L.D.J.K. randomized the participants and performedthe coding of the beverages. M.B.K supervised the study, con-ceived and designed the study, obtained funding and super-vised the development and manufacture of the beverages.M.R.O, L.D.J.K and M.B.K. revised the manuscript critically.

Acknowledgments

We thank the staff, teachers, parents and children of theschools for their willingness to participate and the pleasantcooperation during the study; Emilie de Zoete and HettyGeerars for excellent assistance in the execution of thisstudy; Mr. Joop Bremer, retired head of research of a Dutchsoft drink manufacturer, for his help and advice during devel-opment and manufacture of the beverages; Refresco Beneluxfor meticulous care in producing the beverages; Prof. dr.Jacob Seidell for valuable advice during the study; the mem-bers of our advisory board: Sera de Vries, teacher, Edgar vanMil, MD, Phd, pediatricendocrinologist and Goof Buijs, M.Sc.,

256 J.C. de Ruyter et al. / Contemporary Clinical Trials 33 (2012) 247257senior advisor at Netherlands Institute for Health Promotionand Disease Prevention for creative suggestions; Herbert vanden Heuvel and colleagues, for their dedication in the beveragedistribution; Ellinore Tellegen, for acting as independentphysician.

References

[1] Ogden CL, Carroll MD, Curtin LR, McDowellMA, Tabak CJ, Flegal KM. Prev-alence of overweight and obesity in the United States, 19992004. JAMA2006;295:154955.

[2] Ogden CL, Carroll MD, Curtin LR, Lamb MM, Flegal KM. Prevalence ofhigh body mass index in US children and adolescents, 20072008.JAMA 2010;303:2429.

[3] Hayman LL, Meininger JC, Daniels SR, McCrindle BW, Helden L, Ross J,et al. Primary prevention of cardiovascular disease in nursing practice:focus on children and youth: a scientific statement from the AmericanHeart Association Committee on Atherosclerosis, Hypertension, andObesity in Youth of the Council on Cardiovascular Disease in theYoung, Council on Cardiovascular Nursing, Council on Epidemiologyand Prevention, and Council on Nutrition, Physical Activity, and Metab-olism. Circulation 2007;116:34457.

[4] Hannon TS, Rao G, Arslanian SA. Childhood obesity and type 2 diabetesmellitus. Pediatrics 2005;116:47380.

[5] Steinberger J, Daniels SR. Obesity, insulin resistance, diabetes, and car-diovascular risk in children: an American Heart Association scientificstatement from the Atherosclerosis, Hypertension, and Obesity in theYoung Committee (Council on Cardiovascular Disease in the Young)and the Diabetes Committee (Council on Nutrition, Physical Activity,and Metabolism). Circulation 2003;107:144853.

[6] Magarey AM, Daniels LA, Boulton TJ, Cockington RA. Predicting obesityin early adulthood from childhood and parental obesity. Int J Obes RelatMetab Disord 2003;27:50513.

[7] Lobstein T, Baur L, Uauy R. Obesity in children and young people: a cri-sis in public health. Obes Rev 2004;5(Suppl. 1):482.

[8] Franks PW, Hanson RL, Knowler WC, Sievers ML, Bennett PH, LookerHC. Childhood obesity, other cardiovascular risk factors, and prematuredeath. N Engl J Med 2010;362:48593.

[9] Weiss R, Dziura J, Burgert TS, Tamborlane WV, Taksali SE, Yeckel CW,et al. Obesity and the metabolic syndrome in children and adolescents.N Engl J Med 2004;350:236274.

[10] Duffey KJ, Popkin BM. Shifts in patterns and consumption of beveragesbetween 1965 and 2002. Obesity 2007;15:273947 (Silver Spring).

[11] Nielsen SJ, Popkin BM. Changes in beverage intake between 1977 and2001. Am J Prev Med 2004;27:20510.

[12] Mattes R. Fluid calories and energy balance: the good, the bad, and theuncertain. Physiol Behav 2006;89:6670.

[13] Schulze MB, Manson JE, Ludwig DS, Colditz GA, Stampfer MJ, Willett WC,et al. Sugar-sweetened beverages, weight gain, and incidence of type 2diabetes in young and middle-aged women. JAMA 2004;292:92734.

[14] Palmer JR, Boggs DA, Krishnan S, Hu FB, Singer M, Rosenberg L. Sugar-sweetened beverages and incidence of type 2 diabetes mellitus inAfrican American women. Arch Intern Med 2008;168:148792.

[15] Bes-Rastrollo M, Sanchez-Villegas A, Gomez-Gracia E, Martinez JA,Pajares RM, Martinez-Gonzalez MA. Predictors of weight gain in aMediterranean cohort: the Seguimiento Universidad de Navarra Study1. Am J Clin Nutr 2006;83:36270 quiz 945.

[16] Nicklas TA, Yang SJ, Baranowski T, Zakeri I, Berenson G. Eating patternsand obesity in children. The Bogalusa Heart Study. Am J Prev Med2003;25:916.

[17] Ludwig DS, Peterson KE, Gortmaker SL. Relation between consumptionof sugar-sweetened drinks and childhood obesity: a prospective, obser-vational analysis. Lancet 2001;357:5058.

[18] Dubois L, Farmer A, Girard M, Peterson K. Regular sugar-sweetenedbeverage consumption between meals increases risk of overweightamong preschool-aged children. J Am Diet Assoc 2007;107:92434.

[19] Johnson L, Mander AP, Jones LR, Emmett PM, Jebb SA. Is sugar-sweetened beverage consumption associated with increased fatnessin children? Nutrition 2007;23:55763.

[20] Newby PK, Peterson KE, Berkey CS, Leppert J, Willett WC, Colditz GA.Beverage consumption is not associated with changes in weight andbodymass index among low-income preschool children in North Dakota.J Am Diet Assoc 2004;104:108694.

[21] O'Connor TM, Yang SJ, Nicklas TA. Beverage intake among preschoolchildren and its effect on weight status. Pediatrics 2006;118:e10108.

[22] Vandenbroucke JP. Observational research, randomised trials, and twoviews of medical science. PLoS Med 2008;5:33943.[23] Fewell Z, Davey Smith G, Sterne JAC. The impact of residual and unmea-sured confounding in epidemiologic studies: a simulation study. Am JEpidemiol 2007;166:64655.

[24] DiMeglio DP, Mattes RD. Liquid versus solid carbohydrate: effects onfood intake and body weight. Int J Obes Relat Metab Disord 2000;24:794800.

[25] Tordoff MG, Alleva AM. Effect of drinking soda sweetened with aspar-tame or high-fructose corn syrup on food intake and body weight.Am J Clin Nutr 1990;51:9639.

[26] Raben A, Vasilaras TH, Moller AC, Astrup A. Sucrose compared with ar-tificial sweeteners: different effects on ad libitum food intake and bodyweight after 10 wk of supplementation in overweight subjects. Am JClin Nutr 2002;76:7219.

[27] Chen L, Appel LJ, Loria C, Lin PH, Champagne CM, Elmer PJ, et al. Reduc-tion in consumption of sugar-sweetened beverages is associated withweight loss: the PREMIER trial. Am J Clin Nutr 2009;89:1299306.

[28] James J, Thomas P, Cavan D, Kerr D. Preventing childhood obesity byreducing consumption of carbonated drinks: cluster randomised con-trolled trial. BMJ 2004;328:1237.

[29] Ebbeling CB, FeldmanHA,Osganian SK, Chomitz VR, Ellenbogen SJ, LudwigDS. Effects of decreasing sugar-sweetened beverage consumption on bodyweight in adolescents: a randomized, controlled pilot study. Pediatrics2006;117:67380.

[30] Sichieri R, Paula Trotte A, de Souza RA, Veiga GV. School randomisedtrial on prevention of excessive weight gain by discouraging studentsfrom drinking sodas. Public Health Nutr 2009;12:197202.

[31] Katan MB. Weight-loss diets for the prevention and treatment of obesity.N Engl J Med 2009;360:9235.

[32] Crawford PB, Woodward-Lopez G, Ritchie L, Webb K. How discretion-ary can we be with sweetened beverages for children? J Am DietAssoc 2008;108:14404.

[33] Allison DB, Mattes RD. Nutritively sweetened beverage consumptionand obesity: the need for solid evidence on a fluid issue. JAMA2009;301:31820.

[34] SCF. Sucralose (opinion adapted by the SCF on 7 September 2000).http://ec.europa.eu/food/fs/sc/scf/out68_en.pdfFebruary 19, 2011.

[35] SCF. Re-evaluation of acesulfame K with reference to the previous SCFopinion of 1991 (opinion expressed on 9th March 2000). http://ec.europa.eu/food/fs/sc/scf/out52_en.pdfFebruary 19, 2011.

[36] Snedecor GW, Cochran WG. Statistical methods. 8th ed. Ames, USA:Iowa State University Press; 1989.

[37] Federal Register. National Bureau of Standards. Customary system ofweight and measures. Fed Register 1968;33:345.

[38] Katan MB, Ludwig DS. Extra calories cause weight gainbut how much?Jama 2010 Jan 6;303:656.

[39] Wang YC, Gortmaker SL, Sobol AM, Kuntz KM. Estimating the energygap among US children: a counterfactual approach. Pediatrics2006;118:e172133.

[40] Fredriks AM, van Buuren S, Burgmeijer RJ, Meulmeester JF, Beuker RJ,Brugman E, et al. Continuing positive secular growth change in TheNetherlands 19551997. Pediatr Res 2000;47:31623.

[41] de Jong E, Schokker DF, Visscher TL, Seidell JC, Renders CM. Behaviouraland socio-demographic characteristics of Dutch neighbourhoods with highprevalence of childhood obesity. Int J Pediatr Obes 2011 Aug 6;6:298305.

[42] Cole TJ, Bellizzi MC, Flegal KM, Dietz WH. Establishing a standard defini-tion for child overweight and obesity worldwide: international survey.BMJ 2000;320:12403.

[43] Cole TJ, Flegal KM, Nicholls D, Jackson AA. Body mass index cut offs todefine thinness in children and adolescents: international survey. BMJ2007;28:1667.

[44] CBS. Number of non-western people in the Netherlands. Netherlands.http://statline.cbs.nl/StatWeb/publication/?DM=SLNL&PA=70787NED&D1=a&D2=0&D3=0&D4=a&D5=l&HDR=T,G3&STB=G1,G2,G4&VW=TFebruary 19, 2011.

[45] CBS. Level of education of the Netherlands population (1565 year olds).Netherlands. http://statline.cbs.nl/StatWeb/publication/?DM=SLNL&PA=71822NED&D1=0&D2=0&D3=0&D4=0&D5=0-2,4-10&D6=0&D7=l&HDR=T,G1&STB=G2,G4,G5,G3,G6&VW=T February 19, 2011.

[46] Schulz KF, Grimes DA. Generation of allocation sequences in rando-mised trials: chance, not choice. Lancet 2002;359:5159.

[47] Gibson RS. Principles of nutritional assessment. 2nd ed. New York, USA:Oxford University Press; 2005.

[48] Heymsfield SB, Lohman TG, Wang Z, Going SB. Human body composi-tion 2nd ed. Champaign, USA: Human Kinetics; 2005.

[49] Roberts A, Renwick AG, Sims J, Snodin DJ. Sucralose metabolism andpharmacokinetics in man. Food Chem Toxicol 2000;38(Suppl. 2):S3141.

[50] Garnett SP, Baur LA, Cowell CT. Waist-to-height ratio: a simple optionfor determining excess central adiposity in young people. Int J Obes(Lond) 2008;32:102830.

[51] Deurenberg P, Pieters JJ, Hautvast JG. The assessment of the body fatpercentage by skinfold thickness measurements in childhood andyoung adolescence. Br J Nutr 1990;63:293303.

[52] Rush EC, Puniani K, Valencia ME, Davies PS, Plank LD. Estimation ofbody fatness from body mass index and bioelectrical impedance: com-parison of New Zealand European, Maori and Pacific Island children.Eur J Clin Nutr 2003;57:1394401.

[53] Rosenman K. Benefits of saccharin: a review. Environ Res 1978;15:7081.

[54] Davidson TL, Swithers SE. A Pavlovian approach to the problem of obe-sity. Int J Obes Relat Metab Disord 2004;28:9335.

[55] Swithers SE, Baker CR, Davidson TL. General and persistent effects ofhigh-intensity sweeteners on body weight gain and caloric compensa-tion in rats. Behav Neurosci 2009;123:77280.

[56] Swithers SE, Davidson TL. A role for sweet taste: calorie predictive rela-tions in energy regulation by rats. Behav Neurosci 2008;122:16173.

[57] Rodin J. Comparative effects of fructose, aspartame, glucose, and waterpreloads on calorie and macronutrient intake. Am J Clin Nutr 1990;51:42835.

[58] Black RM, Tanaka P, Leiter LA, Anderson GH. Soft drinks with aspar-tame: effect on subjective hunger, food selection, and food intake ofyoung adult males. Physiol Behav 1991;49:80310.

[59] Ford HE, Peters V, Martin NM, Sleeth ML, Ghatei MA, Frost GS, et al.Effects of oral ingestion of sucralose on gut hormone response andappetite in healthy normal-weight subjects. Eur J Clin Nutr 2011;65:50813.

257J.C. de Ruyter et al. / Contemporary Clinical Trials 33 (2012) 247257

Effect of sugar-sweetened beverages on body weight in children: design and baseline characteristics of the Double-blind, Randomized INtervention study in Kids1. Introduction2. Methods2.1. Outline2.2. DRINK pilot study2.3. Design and setting of main DRINK study2.4. Beverages2.4.1. Formulations2.4.2. Production

2.5. Sample size calculation2.6. Recruitment2.7. Baseline characteristics2.8. Randomization of participants and beverages2.9. Packaging and distribution of study cans2.10. Body measurements2.11. Ancillary measurements2.12. Endpoints2.13. Data analysis and statistics2.14. Timelines

3. Discussion3.1. Strengths and limitations3.2. Interpretation of outcomes

Conflict of interestsAuthor contributionsAcknowledgmentsReferences