The MATADOR (Minimising Adaptive Thermogenesis And Deactivating Obesity Rebound) study was a single-centre, parallel-group, randomised controlled trial. The study was granted ethics approval through the University Human Research Ethics Committee at the Queensland University of Technology, Australia. Fifty-one males with obesity were recruited in cohorts and, after screening, were allocated to the continuous (CON) or intermittent (INT) ER interventions. Participants initially undertook a 4-week baseline (weight stabilisation) phase to determine energy needs and help them to accommodate to the study diet macronutrient composition, and then undertook 16 weeks of ER delivered as either: (1) CON: 16 weeks of continuous (daily) ER, or (2) INT: 16 weeks of ER as 8 × 2-week blocks of ER interspersed with 7 × 2-week blocks of energy balance (30 weeks total; please see study design in Supplementary Item 1). Both groups then completed an 8-week post-weight loss energy balance phase. Including the 4-week baseline, 16- or 30-week ER, and 8-week post-weight loss energy balance phases, the total length of the intervention was 28 and 42 weeks for the CON and INT groups, respectively. Food was provided for each of these three phases (further details in the section Provision of diet, below). Participants were followed up after a 6-month free-living period.

Eligibility criteria for participants

Eligible participants were males aged 25–54 years, with a body mass index classified as obese (30–45 kg m−2), weight-stable (±2 kg for 6 months prior to participation) and sedentary (<60 min of structured moderate to vigorous intensity physical activity per week). Exclusion criteria are listed in Supplementary Item 2.

Recruitment and screening strategies

Supplementary Item 3 is a CONSORT diagram providing an overview of the flow of participants through the study. The three-step screening process to assess eligibility is detailed in Supplementary Item 4. After providing informed consent, participants were randomly assigned in a 1:1 ratio to either the CON or INT interventions. While it was not possible to blind participants or research staff to the assigned treatment groups, there was no discussion with the participants regarding the difference between the two interventions. As shown in the CONSORT diagram, 51 men were randomised and commenced the 4-week baseline weight stabilisation phase, 47 completed the baseline phase, 41 completed the ER phase (16 or 30 weeks, in the CON and INT groups, respectively), and 36 completed the ER phase per protocol, which was defined as completion of the assigned ER intervention (CON or INT) without weight gain while in restriction, and meeting assessment requirements up to and including Week16 ER.

Determination of weight maintenance energy requirements

Weight maintenance energy requirements were estimated for each participant by multiplying measured REE (detailed below) by an appropriate physical activity level based on self-reported work-time and leisure-time physical activity. Participants were prescribed an individualised diet (detailed below) designed to maintain weight stability, and were provided with an electronic weighing scale to self-record body weight at home. These weights were used to assess the adequacy of energy intake for weight maintenance, and to adjust energy intake as required. If participants gained or lost weight consistently over at least 3 days, they were provided with instructions on how to adjust the energy intake of the provided diet to maintain weight stability.

Energy restriction interventions

The study was designed so that the ER diet for participants in both groups was equivalent to 67% of individual weight maintenance energy requirements (that is, 33% reduction in energy intake). The energy intake prescription was adjusted to account for reductions in REE that were measured after every 4 weeks of ER, to ensure that participants remained in the same relative energy deficit throughout the study. Consequently, the absolute deficit (kJ d−1) decreased significantly over time (P<0.001) in both groups, but did not differ between CON and INT groups (P=0.49): WK1-4ER: -4142±442 and −4009±647 kJ d−1; WK5-8ER: −3998±464 and −3885±538 kJ d−1; WK9-12ER: −3902±505 and −3790±583 kJ d−1; WK13-16ER: −3810±533 and −3740±444 kJ d−1. During the seven energy balance blocks in the INT group, participants were prescribed a diet providing 100% of weight maintenance energy requirements.

Provision of diet

Participants were provided with all main meals and morning and afternoon snacks for the duration of the study (28 or 42 weeks for the CON and INT interventions, respectively). Meals were prepared by a commercial kitchen under the direction of a dietician and delivered to the participants’ homes each week. This ‘base’ diet supplied the majority of each participant’s energy requirements. The remaining energy intake came from additional, discretionary items, chosen by individual participants in consultation with a researcher (REW). Inclusion of discretionary items in a long-term (24-–month) dietary intervention has been shown to increase compliance.28 The planned macronutrient distribution in both ER and energy balance diets was 25–30% of energy as fat, 15–20% as protein and 50–60% as carbohydrate. Participants were required to complete daily self-report food diaries for the duration of the study (28 or 42 weeks for CON and INT groups, respectively). The completion of these diaries was required for participants to be considered compliant with the study requirements, but the data have not been analysed as a measure of dietary adherence.

Overview of data collection

As shown in Supplementary Item 1, weight, body composition and REE were measured at the start and end of the 4-week baseline phase, after every 4 weeks of ER, at weeks 1, 2, 4 and 8 of the 8-week post-ER energy balance phase, and at follow-up 6 months later. Resting energy expenditure is not reported for the post-ER energy balance and 6-month follow-up time points due to a large amount of missing data. During ER, measurements were taken after the same number of weeks of ER for both groups. For example, the Week 4 measurement was taken 4 weeks after baseline for the CON group, and 6 weeks after baseline for the INT group (that is, after the first 2 × 2-week blocks of ER separated by 1 × 2-week block of energy balance). As another example, the Week 8 measurement was taken 8 weeks after baseline for the CON group, and 14 weeks after baseline for the INT group (that is, after 4 × 2-week blocks of ER separated by 3 × 2-week blocks of energy balance). Measurements were included every 4 weeks during the ER intervention to provide information on the time course of responses, in addition to pre-post comparisons. All measurements during the ER intervention were taken during restriction in both groups. For the INT group, measurements were made at the end of a 2-week block of ER.

Body height, weight and composition

Height was measured to the nearest 0.1 cm using a Harpenden stadiometer (Holtain Ltd, Crosswell, UK). At each laboratory visit, body weight was measured to the nearest 0.1 kg using a calibrated digital scale, and body composition was calculated from body density measured by air displacement plethysmography (BOD POD, Life Measurement Inc., Concord, CA, USA). In addition to laboratory measurements of weight, all participants were provided with an electronic weighing scale (Model WW147A, Conair Australia, Pty Ltd, Terrey Hills, NSW, Australia), and asked to record body weight at least weekly during the study. These self-reported body weights were used to track progress throughout the study, and provide additional information on the time course of changes in weight in the periods between laboratory visits.

Resting energy expenditure

resting energy expenditure was measured using a ventilated hood system (TrueOne 2400 Metabolic System, ParvoMedics Inc, Sandy, UT, USA), which was calibrated before each measurement using standardised gases. All testing was conducted between 0600 and 0900 h after a minimum 10-h overnight fast. Participants arrived at the laboratory by car and were instructed to minimise physical activity prior to arrival. Testing was performed in a thermo-neutral environment with participants lying supine in a comfortable position, head on a pillow, and a transparent ventilated hood placed over their head. During the measurement period, participants were asked to remain as relaxed as possible without falling asleep, and instructed not to talk or fidget. To reduce boredom and prevent sleep, participants listened to quiet music throughout the measurement. VO 2 and VCO 2 were measured continuously for 30 min. After discarding the first 10 mins of data, REE was calculated as the lowest consecutive 10-min average value, provided that the coefficient of variation within that 10-min interval was <5%. Resting energy expenditure was calculated using the Weir equation.29

Calculation of predicted REE, and of changes from baseline

Given the contention regarding the best analytical approach to assess and define adaptive thermogenesis,30, 31, 32, 33, 34, 35, 36, 37, 38 we examined changes in REE using three approaches:

1 Comparing REE over the intervention after adjustment for changes in fat mass (FM) and fat free mass (FFM). 2 Comparing measured REE with REE predicted from the group-specific equations developed using regression analysis of baseline data (see details below). 3 Comparing measured REE with REE predicted from the reference equation published by Muller et al.39

It has been suggested32 that the most appropriate analysis uses the study-specific regression equation derived from baseline data (REE, body composition, age, sex). As such, linear regression analyses were performed to develop prediction equations for REE from baseline data (group allocation, age, FM, FFM). Whereas age did not significantly explain any of the variance in REE, group allocation accounted for a significant proportion of variance in REE. Consequently, a separate equation was derived for CON and INT:

Given the relatively small, homogeneous sample in the present study, it could be argued that the resulting prediction equation may not be robust.35 To overcome this potential weakness, REE was also predicted from the equation developed by Muller et al.39 on a larger, phenotypically similar cohort (body mass index >30 kgm−2; N=278); where female=0, male=1:

These equations were used to predict REE at baseline, and after 4, 8, 12 and 16 weeks of ER for the CON and INT groups separately. Changes in REE from baseline for measured and predicted values were then compared.

Statistical analyses

All analyses were performed using the STATA statistical software package Version 14.2 (Statacorp LLC, College Station, TX, USA). Data are reported as mean±standard deviation (s.d.) unless otherwise specified. Mixed model repeated measures analyses were employed to determine changes in outcome variables from baseline, and differences between the CON and INT groups accounting for covariates (FM, FFM, age) where appropriate. Linear regression analyses were used to examine relationships between REE and body composition. Differences were considered significant where P<0.05.

Data are reported for all randomised participants via intention-to-treat (ITT) analysis using the Last-Observation-Carried-Forward method, whereby the last available measurement for each participant at the time point prior to withdrawal from the study is retained for each missing time point thereafter in the analysis. The ITT analyses include every participant who was randomised according to randomised treatment assignment, ignoring level of compliance, protocol deviations and withdrawals.40 Consequently, the estimate of treatment effect from ITT analyses is generally conservative. As some participants dropped out during the baseline phase without participating in the ER interventions, analyses are also provided for the sub-samples of all participants who completed the baseline phase (CON=23; INT=24), and those who completed Week 16 of ER (CON=22; INT=19). In this way, we are able to report on the effect of the interventions with the maximum sample at the end of each stage of the study as reported in the CONSORT diagram.

To examine the efficacy of the two interventions, analyses were provided for the cohort of participants who completed Week 16 of ER per protocol (CON=19; INT=17). Finally, as not all participants were available at the 6-month follow-up, analyses are also provided for participants who completed Week 16 of ER per protocol and were available for measurements at the 8-week post-ER energy balance phase and 6-month follow-up (CON=13; INT=15).

The primary outcome variable for the study was weight loss, and the secondary outcome was REE. Over 16 weeks of ER, the energy deficit imposed by a 33% energy deficit would result in an estimated weight loss of ~14 kg for a 110 kg male. However, we and others have found, in well-controlled medium-length (12–24 weeks) dietary restriction interventions, that weight loss is 60–70% of what is predicted from the ER imposed.9, 10, 11 As such, the expected weight loss during continuous ER would be ~9 kg. We tested the hypothesis that weight loss would be greater (higher loss per unit ER) with intermittent vs continuous ER, in part because of attenuation of adaptive thermogenesis (smaller reduction in REE) in response to intermittent ER.

Accounting for the expected variance in starting weight, we calculated that 34 participants (17 per group) would be required, at a statistical power of 0.8 (α=0.05), to detect (two-tailed) a ~5 kg greater weight loss from ER without adaptive thermogenesis (INT: 14 kg) compared with weight loss during continuous ER inducing thermogenic compensation (CON: 9 kg).

It was uncertain a priori the degree to which adaptive thermogenesis could be attenuated using the model of intermittent ER we employed. Body composition-adjusted REE has been shown to decrease by ~400 kJ d−1 within 2 weeks of commencing moderate ER, and larger reductions have been reported in response to more severe and prolonged ER.11, 13, 18, 26, 41 We, and others, have shown the decrease in body composition-adjusted REE, even in response to severe ER, is completely reversed after 10–14 days of reinstating an energy balance diet.11, 20, 41 Consequently, 18 participants (9 per group) would be required at a statistical power of 0.8 (α=0.05) to detect (two-tailed) a 400 kJ d−1 reduction in body composition-adjusted REE compared with no change. Given that REE was measured during ER for both groups, but the magnitude of adaptive thermogenesis was predicted to be modified by the 2-week blocks of energy balance in the INT intervention, we estimated the effect would be potentially halved. To detect a difference of this magnitude (~200 kJ d−1), a cohort of 38 (19 per group) would be required (two-tailed) at a statistical power of 0.8 (α=0.05). Anticipating a 25% drop-out during the intensive intervention, we planned to recruit 50 participants (25 per group).