Study design

This study was conducted as a randomized, parallel arm, controlled, prospective study. The independent variable was nutritional intervention. The primary outcome variables were changes in body composition.

Participants

Figure 1 presents a CONSORT diagram. Twenty-four healthy men with more than 2 years of continuous experience in overload training participated in this randomized controlled study (age = 30 ± 4.5 years; height = 177 ± 3.4 cm; weight = 76.7 ± 5.7 kg; BMI = 23.4 ± 2.2 kg/m2). All volunteered their participation and agreed to complete the supervised training and diet protocols during the 8 weeks of the study. Subjects who had consumed androgenic-anabolic steroids during the last 2 years or those who consumed any type of dietary supplement during the study were excluded. The subjects were advised of the potential risks of the experiment and signed an informed consent form. The study was developed following the ethical guidelines of the Declaration of Helsinki [21]. The investigation was developed in Málaga (Spain). The first evaluation took place on February 2017 and the second measurement on April of the same year.

Procedures

Body composition

Total and regional body composition were estimated using a Hologic QDR 4500 dual-energy x-ray absorptiometry (DXA) scanner (Hologic Inc., Bedford, MA, USA). Each subject was scanned by a certified technician, and the distinguished bone and soft tissue, edge detection, and regional demarcations were done by computer algorithms with APEX Software 3.0 (APEX Corporation Software, Pittsburg, PA, USA). For each scan, subjects wore sport clothes and were asked to remove all materials that could attenuate the X-ray beam, including jewelry items. Calibration of the densitometer was checked daily against standard calibration block supplied by the manufacturer.

Abdominal region was delineated by an upper horizontal border located at half of the distance between acromions and external end of iliac crests, a lower border determined by the external end of iliac crests, and the lateral borders extending to the edge of the abdominal soft tissue. All trunk tissue within this standardized height region was selected for analysis. To determine intertester reliability, two different observers selected the area for each subject manually.

Nutrition intervention

The participants were randomly assigned to a KD group (n = 9), non-KD (NKD) (n = 10) group, and control group (CG) (n = 5). Compliance with the ketosis state was monitored by measuring urinary ketones weekly using reagent strips (Ketostix, Bayer Vital GmbH, Leverkusen, Germany), from week two to the end of the study in KD group. Under the supervision of a registered dietitian, the subjects were given a detailed questionnaire about their work and sociocultural activities, as well as dietary preferences in order to estimate the basal metabolic rate and physical activity-related energy expenditure. Subjects were classified as active in their day-to-day lives, estimating total energy expenditure in line with the indications [22]. Once energy expenditure was determined, together with their weekly training load, a moderate energy surplus was established for experimental groups, since it has been noted that trained men do not require energy increases as high as novice subjects [23, 24]. To guarantee a hyperenergetic condition, a daily energy intake of ≈39 kcal·kg− 1·d− 1 was used in all subjects. To ensure a maximal anabolic response, NKD group was given a protein intake of 2 g⋅kg− 1⋅d− 1, as it is recommended for building muscle mass in trained subjects [2, 22, 25], while 25% of total energy intake corresponded to fat and the remaining calories were given in carbohydrates. Macronutrient distribution for NKD group was about 55% CHO; 20% PRO and 25% FAT. On the other hand, ≈42 g total carbohydrates per day were administered to KD group to ensure the ketosis state [26, 27]. Protein intake was 2 g⋅kg− 1⋅d− 1, and the remaining calories were given in fat with a estimating of 3.2 g∙kg− 1⋅d− 1. Macronutrient distribution for KD group was about <10% CHO; 20% PRO and 70% FAT. Ad libitum meal timing and frequency throughout the day was allowed to improve dietary adherence. Even though a specific number of meals per day is not necessary, provided the daily energy intake is guaranteed [22], from 3 to 6 meals were recommended, with the respective foods selected for the KD group.

Training protocol

During 8 weeks both KD and NKD groups completed four sessions per week of a hypertrophy training protocol, organized into a 2-days upper- and 2-days lower-limb, with 72 h of rest between sessions to encourage recovery [28] (Fig. 2).

Fig. 2 Overview of training protocol. WK: Workout (microcycle); UL: Upper-Limb; LL: Lower-Limb; R: Rest; 30X: 3 s of eccentric contraction and explosive movement during concentric activity Full size image

Participants were experienced in overload training and used to different nutritional strategies; therefore, no familiarization session was necessary. Moderate to high loads were used to encourage mechanical tension [29]. Rest between sets lasted 3 min, so that volume did not decline [30, 31]. Cadences were explosive in the concentric activation, and 3 s long during the eccentric contraction to generate more muscle damage [29, 32]. Two weekly stimuli were provided for each muscle group in order to optimize the final results [33]. Push and pull exercises were interspersed for better recovery [34]. Subjects from both groups were asked to increase loads as long as they exceeded repetition rates and had no error technique. During the intervention, all participants were monitored by an RT specialist who supervised and checked the load at each training session, and made the relevant adjustments when was necessary. Meanwhile, men in control group were asked to maintain their current level of physical activity during the study.

Statistical analysis

Descriptive statistics tests were applied (mean and standard deviation, SD). Data were analyzed using a univariate, multivariate and repeated measures general linear model (GLM), with two levels by time (pre- and post-test) and considering groups (KD, NKD and CG) as inter-subjects factor. Wilks’ Lambda multivariate tests are reported to describe overall effects of related variables analyzed. Greenhouse-Geisser univariate tests with least significant difference and post-hoc comparisons (Bonferroni correction) are presented for individual variables analyzed. Partial eta squared effect sizes (ηp2) were also reported on select variables as an indicator of effect size (ES) of the repeated measures GLM. An Eta squared around 0.02 was considered small, 0.13 medium, and 0.26 large [35]. Furthermore, one-way analysis of variance (ANOVA), with a 95.0% confidence level and Bonferroni post-hoc correction, as is recommended for these studies [36, 37], was performed to detect between-group differences in the Δ changes (post-test – pre-test). In addition, ES calculation was done with Cohen’s d, as a standardized measurement based on SD differences; while d = 0.2 was considered a small effect, d = 0.5 was a medium effect and d = 0.8 was a large effect, which is used as a guide for substantive significance. The normal Gaussian distribution of the data was verified by the Shapiro-Wilk test. Mean changes with 95% CI’s completely above or below baseline are considered significant changes from baseline. These statistical analyses were performed with licensed Statistical Package for the Social Sciences (SPSS) software (SPSS 24.0, SPSS Inc., Chicago, USA) and GraphPad software (GraphPad Prism 7.03, California, USA).