Participants, Randomization, and Blinding
This investigation was part of a larger RCT performed between February 2014 to July 2016 at the Exercise and Health Laboratory, Faculty of Human Kinetics, University of Lisbon, and was carried out following the recommendations of the Declaration of Helsinki for Human Studies. The protocol was approved by the Ethics Committee of the Portuguese Diabetes Association (approval number: 07/17/2013). Written informed consents were obtained from all participants.
The complete study protocol has been previously published (22). Briefly, individuals with T2DM were recruited to analyse the impact of a 1-year exercise intervention with different intensities on glycated haemoglobin (HbA1c) (clinicaltrials.gov ID:NCT03144505). Eighty patients were recruited within the Lisbon Metropolitan Area (Figure 1) (22), and posteriorly randomized into three groups (control, HIIT with RT or MCT with RT). An external researcher, with an allocation ratio of 1:1:1, used a computer-generated list of random numbers, for the randomization process. The researchers performing the assessments were blinded to group randomization. Inclusion criteria for participants included adults diagnosed with T2DM (23), age between 30-75 years old, no major micro or macro vascular complications from diabetes, body mass index <48 kg/m2, and no limitations that would prevent them from practicing exercise. The main outcome power and sample size calculations (G-Power, Version 3.1.3) were based on a predicted HbA1c difference of 0.66% with a SD of 1.2%, α=0.05, 1-β=0,80 and an expected dropout rate of 10% (24). For this analysis, the power and sample size calculations were based on changes in whole-body fat, which is related with overall inflammatory profile. Given a predicted whole-body fat difference of 2.7%, with a SD of 1.7%, α=0.05, 1-β=0.80, the sample used on this study was powered for this analysis (16).
The control group had an initial standard physical activity (PA) recommendation session and no structured exercise. All of the exercise groups (i.e. the MCT and HIIT group) had three supervised exercise sessions per week, monitored with a heart rate polar band (Polar T-31, USA). The exercise programs of both groups were developed to have matched energy expenditure, with a weekly target of 10 kcal/kg, which was updated monthly for their body weight and every 3 months for their peak oxygen consumption.
The MCT and HIIT groups had an exercise periodization for the 1-year divided in two and three phases, respectively. Heart rate reserve (HRR), calculated through the Karvonen formula (25), was used to achieve prescribed intensities. Phase 1 was identical for both groups (weeks 1–4), with patients performing continuous cycling of moderate-intensity (40-60% of the HRR) with durations increasing from 15 minutes to 25 by the end of week 4. The MCT group had only one additional phase (training phase, weeks 5-52), where participants exercised at 40 to 60% of the HRR, with durations based on prescribed energy expenditure targets.
In the HIIT group, during phase 2 (5-8 weeks), patients performed bouts of 2 minutes of cycling at 70% of the HRR followed by 1 minute at 40-60% of the HRR (weeks 5-6), and increased to bouts of 80% (1.5 minutes) of the HRR followed by 1 minute at 40-60% of the HRR (weeks 7-8), while maintaining energy expenditure targets. In phase 3 (weeks 9-52), participants in the HIIT group performed 1 minute of exercise at 90% of their HRR followed by 1 minute resting at 40-60% of the HRR. Both the MCT and HIIT group were further complemented with a whole-body RT, after the aerobic component, which included 1 set of 10-12 RM of eight exercises (seated row, pulldown, chest press, shoulder press, leg press, one leg lung, dead bug and regular plank).
Anthropometry and Body Composition
Patients were weighed on an electronic scale, to the nearest 0.01 kg while wearing minimal clothes (Seca, Hamburg, Germany). Height was measured to the nearest 0.1 cm with a stadiometer (Seca, Hamburg, Germany). Waist circumference was taken according to the standardized procedures of the National Institute of Health (26).
Dual energy X-ray absorptiometry (Hologic Explorer-W, Waltham, USA) was used to assess regional and total body fat, following standardized protocols and procedures set out by the manufacturer. Whole-body fat index (WBFI) and abdominal fat index (AFI) were calculated by dividing the total and abdominal fat mass by the square of the height (kg/m2).
Objective Measures of Moderate-to-Vigorous Physical Activity
Moderate-to-vigorous PA (MVPA) was assessed by accelerometry (ActiGraph, GT3X+, FL, USA) at baseline prior to the start of the intervention. All participants used the accelerometer for 7 days, on the right hip. The devices were activated on raw mode with a 100 Hz frequency and later transformed into 15-s epochs. The Troiano et al. (27) cut points and wear time validation criteria were used.
Laboratory Measurements
Blood collection was performed in a seated position from the antecubital vein at rest after an overnight fast into dry tubes and into tubes containing ethylenediamine-tetraacetic acid as an anticoagulant. Biological samples were centrifuged at 500 g at 4 °C for 15-min and plasma samples were frozen at -80 °C for posterior analysis.
Serum samples were used to analyse the lipid profile of the participants, including the quantification of total cholesterol, LDL-C and HDL-C cholesterol, and triglycerides using colored enzymatic tests in an automated analyser (auto analyser Olympus AU640, Beckman Coulter). Plasma samples were then used for TNF- α, IL-6, sCD163, C-reactive protein (CRP), and cortisol quantification using commercial ELISA kits (DiaSource Immuno Assays S.A for TNF- α, IL6, and Cortisol; IBL International GMBH for CRP; and DC1630, R&D Systems for sCD163).
Changes in the lipid and inflammatory profile were analyzed at baseline and at the 1-year follow-up.
Statistical Analysis
Data analyses were performed using SPSS Statistics version 22.0 (SPSS Inc., an IBM Company, Chicago, Illinois, USA). Results are presented as means ± SD for all normally distributed outcomes and as median and inter-quartile range for skewed outcomes. Comparisons between groups were performed using the Chi-squared test for sex proportions between groups, and the parametric independent sample ANOVA test with a Bonferroni post-hoc analysis for normally distributed variables or the non-parametric Kruskal–Wallis test in absence of normality.
Between-group and within-group effects for the lipid inflammatory profile were performed using generalized estimating equations followed by a least significant difference post hoc test. Models were adjusted for potential confounders (i.e. age, sex and baseline MVPA). All the outcomes went through an intention-to-treat analysis (ITTA). An additional per-protocol analysis (PPA) was performed in only those who completed both assessments (i.e. baseline and 1-year), had at least 70% attendance to all the exercise sessions, and in those without substantial changes in pharmacological therapy (22). No changes were made to dyslipidemia and hypertension medication, however, individuals with major changes in anti-hyperglycemic medication, such as transitioning to insulin, were removed from the (PPA).