Very low-carbohydrate high-fat diet improves risk markers for cardiometabolic health more than exercise in men and women with overfat constitution: Secondary analysis of a randomized controlled clinical trial

ORIGINAL RESEARCH published: 23 May 2022 doi: 10.3389/fnut.2022.867690 Frontiers in Nutrition | www.frontiersin.org 1May 2022 | Volume 9 | Article 867690 Edited by: Daniel Moore, University of Toronto, Canada Reviewed by: Stefan Kabisch, Charité Universitätsmedizin Berlin, Germany Hidetaka Hamasaki, Hamasaki Clinic, Japan Jonathan Peter Little, University of British Columbia, Canada *Correspondence: Lukas Cipryan [email protected] orcid.org/0000-0002-2403-8797 Specialty section: This article was submitted to Sport and Exercise Nutrition, a section of the journal Frontiers in Nutrition Received: 01 February 2022 Accepted: 29 April 2022 Published: 23 May 2022 Citation: Cipryan L, Litschmannova M, Maffetone PB, Plews DJ, Dostal T, Hofmann P and Laursen PB (2022) Very Low-Carbohydrate High-Fat Diet Improves Risk Markers for Cardiometabolic Health More Than Exercise in Men and Women With Overfat Constitution: Secondary Analysis of a Randomized Controlled Clinical Trial. Front. Nutr. 9:867690. doi: 10.3389/fnut.2022.867690 Very Low-Carbohydrate High-Fat Diet Improves Risk Markers for Cardiometabolic Health More Than Exercise in Men and Women With Overfat Constitution: Secondary Analysis of a Randomized Controlled Clinical Trial Lukas Cipryan1*, Martina Litschmannova2, Philip B. Maffetone3, Daniel J. Plews4, Tomas Dostal1, Peter Hofmann5and Paul B. Laursen4 1Department of Human Movement Studies & Human Motion Diagnostic Centre, The University of Ostrava, Ostrava, Czechia, 2Department of Applied Mathematics, VSB—Technical University of Ostrava, Ostrava, Czechia, 3Independent Researcher, Brattleboro, VT, United States, 4Sports Performance Research Institute New Zealand (SPRINZ), Auckland University of Technology, Auckland, New Zealand, 5Institute of Human Movement Science, Sport & Health, Exercise Physiology, Training & Training Therapy Research Group, University of Graz, Graz, Austria Purpose: This randomized controlled parallel-group study examined the effects of a very low-carbohydrate high-fat (VLCHF) diet and high-intensity interval training (HIIT) program over 12-weeks on cardiometabolic risk factors in individuals with overfat constitution. Methods: Ninety-one participants out of 109 completed the study. The participants were randomly allocated to the HIIT (N=22), VLCHF (N=25), VLCHF+HIIT (N=25), or control (N=19) groups for 12 weeks. Fasting plasma samples were collected before the intervention and after 4 and 12 weeks. The analyzed outcomes included complete blood count, glucose, insulin, glycated hemoglobin, triglycerides (TG), cholesterol, highand low-density lipoprotein (HDL-C and LDL-C), lipoprotein(a), adiponectin (Adpn), leptin (Lep), tumor necrosis factor α(TNF-α), other interleukins (hs-IL-6, IL-1β, and IL-10), and IL-1RA. The homeostasis model assessment of insulin resistance (HOMA-IR), Adpn/Lep ratio, TG/HDL-C ratio, and TyG index were calculated and analyzed. Blood pressure was measured before the intervention, after 4, 8, and 12 weeks (ClinicalTrials.gov: NCT03934476). Results: Absolute changes in HOMA-IR, Adpn/Lep ratio, LDL-C, and diastolic blood pressure after 12 weeks differed by study groups (p<0.05). The most pronounced changes were revealed in the VLCHF (1M [95% CI]; HOMA-IR: −0.75 [−1.13; −0.55]; Adpn/Lep: 9.34 [6.33; 37.39]; LDL-C: 0.06 [−0.12; 0.50] mmol/l) and VLCHF+HIIT (HOMA-IR: −0.44 [−1.14; 0.12]; Adpn/Lep: 4.26 [2.24; 13.16]; LDL-C: 0.25 [−0.04; 0.50] mmol/l) groups. Cipryan et al. VLCHF Diet and HIIT Conclusions: A 12-week VLCHF diet intervention in individuals with overfat constitution is effective for favorable changes in HOMA-IR (compared to HIIT), Adpn/Lep ratio, and diastolic blood pressure. HIIT, or HIIT combined with the VLCHF diet, had no additional benefits for the analyzed variables. No adverse side effects were observed. Keywords: HOMA-IR, adiponectin, leptin, TG/HDL-C, TyG index, low carbohydrate diet, exercise, overfat INTRODUCTION Physical activity levels in the Western population have not reduced and changed little, despite dramatic increases in the overfat pandemic of the past 30-plus years. More than half of US adults meet the federal 2008 Physical Activity Guidelines for Americans and regular exercise using either aerobic or musclestrengthening exercise, increasing from 44% in 1998 to almost 52% in 2014 (1). Those meeting these guidelines for aerobic activity and muscle-strengthening exercise also increased from about 14% in 1998 to 21% in 2014. However, rates of adults with overweight or obesity rose to almost 71% during a similar period, reflecting the overfat prevalence increase from 75% to over 90% (2). Cardiovascular and metabolic (cardiometabolic) risk factors can contribute significantly to increased morbidity and mortality, reduced quality of life, and higher healthcare costs. Three of the major risk factors include excess body fat, low-grade systemic chronic inflammation, and insulin resistance (IR). Overfat is defined as excess body fat that impairs health (3). Determination of overweight and obese classifications are traditionally based on measures of body mass index (BMI). This is not a direct measure of body fat and can misclassify up to 50 % or more patients with both increased body fat and its associated disease risk factors. Therefore, body fat needs to be considered directly to assess a high-risk body composition (4). Overfat, and its downstream IR and chronic inflammation, which can maintain a diet-induced viscous cycle, can also lead to a wide range of cardiometabolic health problems such as metabolic syndrome, atherosclerosis, hypertension, dyslipidemia, and advanced chronic conditions such as Type 2 diabetes, cardiovascular diseases, cancers, and neurodegenerative diseases (2). In addition, fasting triglycerides and highdensity lipoprotein cholesterol, particularly the ratio of these two measures (TG/HDL-C), are also considered a significant cardiometabolic risk factor as the ratio reflects IR (5). The TG/HDL-C ratio may be a better clinical screening index than the homeostasis model assessment of IR (HOMA-IR) due to accessibility, reproducibility, and cost, and is already a commonly used measure in clinical practice. Exercise and diet are two commonly used modifiable lifestyle factors that can help reduce cardiometabolic risk factors to influence morbidity and mortality, improve quality of life, and reduce healthcare costs. While the importance of physical activity for increased fitness is undeniable, exercise alone may not necessarily reduce excess body fat. In a previous randomized controlled clinical trial we showed that a very lowcarbohydrate high-fat (VLCHF) diet alone reduced excess body fat in individuals with overfat constitution more than highintensity interval training (HIIT) alone (6). Low carbohydrate diets are effective in remission of diabetes (7) and improve insulin sensitivity as measured by HOMA-IR (8–10). Similarly, insulin sensitivity is also related to the degree of physical activity. Exercise has been shown to ameliorate insulin action in insulin-resistant individuals (11) by improvement of the pathophysiologic pathways involved in insulin resistance (12). The beneficial exercise effect on insulin sensitivity occurs after several weeks (13) or even after a single bout of exercise in adults with obesity (14). Moreover, it seems that HIIT induces similar acute improvements in peripheral insulin sensitivity as moderate-intensity continuous training (15). A VLCHF diet was previously shown to increase adiponectin/leptin ratio reflecting reduced systemic lowgrade inflammation in healthy young individuals (16). In contrast, the same diet was found to be an effective strategy for reducing excess body fat in men and women with overfat constitution (6). Systematic reviews and meta-analyses show also beneficial effects of low carbohydrate diets combined with exercise on body composition, triglycerides, and aerobic capacity in adults with obesity (17,18). Consuming a high fat diet, especially one with high saturated fatty acids (SFA), is thought to impair key aspects of cardiometabolic health. This is despite no evidence-based associations between high intake of SFA and risk of atherosclerotic progression (19). Therefore, the purpose of this randomized controlled parallel-group study is to examine the effects of a 12-week VLCHF diet and high-intensity interval training (HIIT) program on cardiometabolic risk factors in men and women with overfat constitution aged 20–59 years. METHODS Parent Study It was a randomized, controlled, four-arm, parallel exercise and/or dietary intervention study (ClinicalTrials.gov: NCT03934476), with the primary aim of examining the VLCHF and HIIT effect on body composition and cardiorespiratory fitness level (6). The method for random assignment is presented in Supplemental Material. There were 91 participants allocated to the four study groups and these completed a 12-week experimental period (Figure 1). Participants were randomly allocated to four study groups: 1) high-intensity interval training (HIIT) and habitual diet, 2) very low-carbohydrate, high-fat diet (VLCHF) and habitual physical activity (no regular exercise training), 3) VLCHF diet and HIIT, and 4) Control (habitual diet and physical activity, no regular exercise training). Dualenergy X-ray absorptiometry (DXA) and graded exercise test to Frontiers in Nutrition | www.frontiersin.org 2May 2022 | Volume 9 | Article 867690 Cipryan et al. VLCHF Diet and HIIT FIGURE 1 | Flow chart (6). Bold value indicates number of participants. Frontiers in Nutrition | www.frontiersin.org 3May 2022 | Volume 9 | Article 867690 Cipryan et al. VLCHF Diet and HIIT volitional exhaustion were used for the body composition and cardiorespiratory fitness (CRF) assessments, respectively. To obtain measures of no intervention, a control group was utilized. Participants in the control group were advised not to change their habitual diet and physical activity regime. Therefore, no diet advice was provided. Results for the primary outcome were previously reported, that a VLCHF diet, either in isolation or in combination with HIIT, caused a significant reduction in visceral adipose tissue (VAT) mass and body composition variables. HIIT alone did not induce such effects on body composition, but improved exercise capacity (6). We utilized the infrastructure of this trial to conduct a preplanned ancillary study focused on clinically relevant risk factors of cardiometabolic health. We analyzed blood samples following a 3-h fast before the experimental period (T0) and after 4, and 12 weeks (T1and T3). Blood pressure was analyzed also after 8 weeks (T2). The participant set used for the primary analysis was identical with the participant set presented in this study. Participants We enrolled adults aged 20–59 years with BMI 25.00–40.00 kg/m2, who were not engaged in any regular exercise. Participants with known chronic diseases were excluded. Additional eligibility criteria are listed in Supplementary Table 1. The participants had no previous experience with the VLCHF diet or HIIT. The recruitment details and dropouts during the study are shown in Figure 1. Written informed consent was obtained from all study participants. The study design was approved by the Ostrava university Ethics Committee (nr. 1/2018). High-Intensity Interval Training (HIIT) Prior to the intervention, the participants were provided with detailed instructions on the HIIT program. This was for both the HIIT and VLCHF+HIIT groups and done both in verbally and written form. The participants were instructed to complete 3 HIIT sessions per week where one HIIT session was completed during weeks 4, 8, and 12 when the participants visited the laboratory and two were home-based and self-performed. Each HIIT session had a warm up and cool down period of 5-min of slow walking. HIIT consisted of a 3 min interval of high-intensity walking (Borg’s scale RPE 18–19) followed by a 3 min interval of low-intensity walking (RPE 9–11). Participants performed 4, 6, and 8 high-intensity intervals in the first, second, and third 4week period, respectively. Therefore, duration at high-intensity was 12, 18 and 24 min and total session time increased from 31 to 43 min and 55 min during each 4-weeks, respectively. Training intensity was measured with a heart rate monitor (Polar M430; Polar Electro, Oy, Finland). These data were subsequently uploaded to Polar Flow (Polar Electro, Finland) and analyzed regularly to track compliance. Participants were instructed to record all additional training sessions of any type in addition to the study protocol. Dietary Intervention Both the HIIT and control groups were asked to maintain their habitual dietary intake without restriction. The VLCHF diet was defined as allowing no more than 50 g of carbohydrates (CHO) per day (20). Neither diet included a specific calorie or energy goal. However, participants in the VLCHF group were advised to compensate for the total energy decrease caused by CHO intake restriction by increasing their natural non-trans-fat intake (e.g., cream, butter, olive, and coconut oil). A target protein intake of 1.5 g/kg lean body mass was recommended. Contrary to the strict CHO restriction, participants were asked to keep to targets. The use of all sweetened and grain-based products had to be minimized. The recommended food included whole food sources, such as meats, vegetables, non-sweetened products, fullfat dairy items, nuts, and seeds. A dietitian provided detailed dietary advice before and during the study (on request or at least once a month). In addition, a handbook was provided to participants containing food lists, guidelines for estimating macronutrient amounts, and sample recipes. To record all foods and quantities consumed an app was used in all study groups (www.kaloricketabulky.cz). This commenced seven days before the start of the intervention. Alcoholic beverages were restricted during the intervention period, and dietary supplements were not permitted 1 month before and during the intervention period. Caffeinated beverages were restricted only before the laboratory sessions. Anthropometric Analysis The results of the anthropometric analysis have previously been reported (6). In summary, the total body mass and visceral adipose tissue (VAT) mass significantly decreased in the VLCHF (by median [IQR]: −6.9 ([−8.4; −5.6]) % and −23.2 [−26.5; −14.7] %, respectively) and VLCHF+HIIT (by −9.0 [–10.9; −7.9] % and −17.6 [−23.8; −10.8] %, respectively) groups after 12 weeks despite no significant changes in the HIIT and Control groups. Laboratory Methods Fasting blood samples were collected from the antecubital vein. Whole blood samples with EDTA as an anticoagulant were used immediately for blood count and HbA1c determination. Serum collection tubes were allowed to clot for 30 min and subsequently centrifuged at 2 500 g for 10 min to separate the serum. Blood serum was divided into three 1-ml aliquots, which were frozen at −80◦C until analysis. The S-Monovette R system (Sarstedt, Nümbrecht, Germany) was used for blood sample collection. Blood count parameters were measured using a UniCel R  DxHTM 800 hematology analyzer (Beckman Coulter, Inc., Brea, CA, USA). Glycated hemoglobin (HbA1c) was measured using a D-10TM Bio-Rad device (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Glucose, triglyceride (TG), total cholesterol, and highand low-density lipoprotein cholesterol (HDL-C and LDLC, respectively) concentrations were measured using an AU 5820 device (Beckman Coulter, Inc., Brea, CA, USA). Serum levels of leptin, adiponectin, TNF-α, IL−1RA, IL−1β, and IL10 were determined by multiplex technology using a BioPlex MAGPIX system (Bio-Rad Laboratories, Redmond, WA, USA). Hs-IL-6 concentrations were measured using a Human IL-6 Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) on a DSX device (Dynex Technologies, Chantilly, VA, Frontiers in Nutrition | www.frontiersin.org 4May 2022 | Volume 9 | Article 867690 Cipryan et al. VLCHF Diet and HIIT USA). Insulin concentration was measured using a UniCel DxI 800 analyzer (Beckman Coulter, Inc., Brea, CA, USA). Lipoprotein(a) [Lp(a)] concentration was measured using a BN ProSpec system (BN ProSpec, Siemens Healthcare Diagnostics Product GmbH, Germany). The intra-assay coefficients of variation for biochemical and blood count parameters were <5%. Leptin, adiponectin, TNF-α, IL−1RA, IL−1β, IL−10, and hs-IL−6 were determined with an inter-assay coefficient lower than 10%. Triglyceride-glucose (TyG) index was calculated by applying the following equation (21): TyGindex =ln fasting serum TG ×fasting plasma glucose 2(1) Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated using the formula (22): HOMA −IR =plasma glucose ×serum insulin 22.5 (2) A capillary blood sample was drawn from a finger to measure β-hydroxybutyrate (βHB) (FreeStyle Optium Neo, Oxon, United Kingdom). All participants self-analyzed it twice a week (every Monday and Thursday) in a fasting state in the morning to monitor responses to the VLCHF diet and to control for adherence. Blood Pressure Systolic and diastolic blood pressure (BP) was automatically measured three times with 1–2 min apart after the participant had been sitting for ≥10 min in a quiet room by applying a standard device (Nissei DM 3000, Nihon Seimitsu Sokki Co., Japan). This procedure is in line with the recommendations of the American College of Cardiology, American Heart Association, and European Society of Hypertension (23,24). Participants were instructed to avoid caffeinated beverages for at least 60 min before the blood pressure measurements. Statistical Analyses The categorical variable (sex) was described by frequency ratio, and numerical variables were described by median and interquartile range (IQR) at each time point. Subsequently, the absolute changes of the monitored variables at time T1, T2, and T3with respect to the baseline (T0) were analyzed. The absolute changes were tested for normal distribution using the Shapiro-Wilk test. In some cases, significant deviations from normality were detected such that non-parametric methods of data description (median and interquartile range) and statistical inference were used. Significance of change was tested by 95% confidence interval (CI) of median and two-tailed Wilcoxon signed-rank test for each variable, each group, and each time. The effect size (ES) of the observed changes was specified by the Wilcoxon effect size (r), including its 95% confidence interval. Threshold values for ES were 0.10 to <0.30 (small), 0.30 to < 0.50 (medium), ≥0.50 (large). Finally, the absolute changes in the given variables for the HIIT, VLCHF, HIIT+VLCHF, and Control groups were compared using the Kruskal-Wallis test at each time point. Dunn’s test was used to analyze specific sample pairs for stochastic dominance. Dunn’s test multiple comparison p-values was adjusted with the Benjamini-Hochberg method. The effect size of the observed differences was assessed using the eta squared based on the H-statistic, including its 95% confidence interval. Threshold values for ES eta squared were 0.01 to <0.08 (small), 0.08 to <0.26 (medium), ≥0.26 (large) (25). An a priori power analysis using GPOWER (26) with power set at 0.80 and significance level set at 0.05 was calculated retrospectively. The power analysis indicated that a total sample of 76 people would be needed to detect large effects (f=0.40) for this study with 4 groups. A total sample of 180 people would be needed to detect medium effects (f=0.25) (27). Thus, the sample size was sufficient to reveal that a large effect could not be interpreted as non-significant. In all cases, statistical significance was set at p<0.05. Statistical analyses were performed using R Core Team (28). RESULTS Participants The flow chart of participants through the trial, as well as the reasons of dropouts, are depicted in Figure 1. Participant characteristics at baseline are listed in Table 1. Diet Total energy intake decreased (p<0.05) in the HIIT (median [95% CI]: −6.1 [−0.2; −13.4] %), VLCHF (−19.7 [−12.5; −25.2] %), and VLCHF+HIIT (−25.8 [−20.5; −28.0] %) groups. Carbohydrate intake decreased (p<0.05) by −81.8 [−79.1; −82.9] % and −82.8 [−80.4; −85.7] % in the VLCHF and VLCHF+HIIT groups, respectively. Fat intake increased by 44.6 [36.1; 61.7] % and 34.8 [24.6; 47.3] % in the VLCHF and VLCHF+HIIT groups, respectively. Protein intake did not significantly change in any of the study groups. Total energy, protein, and carbohydrate intake did not significantly change in the control group, whereas fat intake decreased (p=0.023, −6.0 [−1.0; −17.5] %) (Supplementary Table 2). High-Intensity Interval Training There were substantial between-group differences in the training characteristics. Total training time in the HIIT and VLCHF+HIIT groups (median 1424 and 1452 min, respectively) was substantially higher than those without the HIIT intervention (VLCHF−124 min, Control−105 min). A detailed training session analysis has already been published in the parent study (6). Biochemical Analysis There were no significant between-group differences in all the biochemical variables at baseline. Absolute changes in HOMA-IR after 12 weeks differed by study group (p=0.013; ES 95% CI: small to large). However, no intervention group significantly differed from the Control Frontiers in Nutrition | www.frontiersin.org 5May 2022 | Volume 9 | Article 867690 Cipryan et al. VLCHF Diet and HIIT TABLE 1 | Baseline characteristics of study participants (all Caucasian). HIIT (N=22) VLCHF (N=25) VLCHF+HIIT (N=25) Control (N=19) Between-group differences M(IQR) M(IQR) M(IQR) M(IQR) p-value Male:Female 6:16 8:17 7:18 6:13 0.648 Age (year) 46 (38.8; 53.3) 43 (35.0; 51.5) 43 (32,51) 40 (31; 53) 0.722 Height (cm) 167.7 (160.6; 175.0) 170.1 (164.7; 177.9) 169.8 (160.9; 179.7) 171.9 (162.8; 177.6) 0.336 BMI 28.7 (26.9; 30.9) 31.3 (27.7; 33.0) 31.0 (27.2; 35.1) 28.7 (26.7; 32.9) 0.690 WHtR 0.60 (0.54; 0.62) 0.62 (0.56; 0.65) 0.62 (0.54; 0.68) 0.60 (0.54; 0.63) 0.447 Systolic BP (mmHg) 127 (114; 137) 132 (119; 144) 128 (121; 138) 124 (121; 134) 0.105 Diastolic BP (mmHg) 77 (72; 87) 88 (77; 93) 85 (80; 91) 82 (73; 88) 0.919 Hemoglobin (g/l) 137 (135; 145) 140 (132; 150) 140 (130; 148) 137 (130; 150) 0.934 Hematocrit (%) 0.406 (0.396; 0.421) 0.411 (0.389; 0.441) 0.41 (0.378; 0.433) 0.405 (0.381; 0.435) 0.969 Erythrocytes (1012/l) 4.57 (4.4; 4.81) 4.72 (4.29; 5.07) 4.62 (4.36; 4.91) 4.56 (4.44; 4.88) 0.789 Thrombocytes (109/l) 244 (217; 294) 241 (210; 274) 234 (200; 267) 239 (202; 280) 0.623 Leukocytes (109/l) 6.5 (5.9; 7.6) 6.5 (5.9; 7.9) 6.4 (5.3; 7) 6.7 (5.4; 7.6) 0.466 HbA1c (mmol/mol) 34 (31.5; 38.5) 34 (32; 37.8) 36 (34; 38) 34 (30; 37) 0.300 Glucose (mmol/l) 5.17 (4.94; 5.4) 5.21 (5.01; 5.59) 5.12 (4.42; 5.34) 4.91 (4.65; 5.43) 0.578 Triglycerides (mmol/l) 1.18 (0.86; 1.81) 1.28 (0.86; 2.23) 0.96 (0.77; 2.18) 1.68 (0.79; 2.32) 0.410 Cholesterol (mmol/l) 5.21 (4.51; 5.93) 5.55 (5.15; 6.14) 5.11 (4.39; 6.05) 5.47 (4.91; 6.16) 0.764 HDL-C (mmol/l) 1.36 (1.17; 1.55) 1.29 (1.04; 1.53) 1.18 (1.07; 1.47) 1.32 (1.02; 1.57) 0.210 LDL-C (mmol/l) 3.3 (2.61; 3.76) 3.49 (3.03; 3.9) 3.16 (2.49; 3.59) 3.47 (2.95; 3.79) 0.386 Insulin (mU/l) 7.7 (5.2; 10.2) 9.6 (6.2; 14.6) 7.3 (5.3; 12.2) 8.2 (7.3; 14.1) 0.436 Leptin (ng/l) 5.7 (2.8; 7.4) 6.9 (3.4; 12.9) 6.2 (4.6; 12.6) 6.2 (3; 12.3) 0.579 Adiponectin (mg/l) 27 (21; 39) 41 (22; 59) 42 (16; 58) 27 (15; 59) 0.735 TG/HDL-C (–) 0.77 (0.56; 1.37) 1.07 (0.6; 2.05) 0.74 (0.55; 1.9) 1.15 (0.47; 2.32) 0.585 TyG index (–) 3.06 (2.25; 4.67) 3.24 (2.32; 5.9) 2.63 (1.86; 5.68) 4 (2.15; 5.4) 0.398 HOMA-IR (–) 1.78 (1.13; 2.5) 2.03 (1.44; 3.66) 1.51 (1.13; 3.11) 1.92 (1.51; 3.51) 0.726 Adpn/Lep (–) 5.66 (3.39; 9.98) 5.06 (3.29; 9.53) 4.19 (2.53; 9.61) 7.16 (2.24; 22.11) 0.648 Legend: BMI, body mass index; WHtR, waist-to-height ratio; BP, blood pressure; HbA1c, glycated haemoglobin; TG, triglycerides; HDL-C/LDL-C, high/low density lipoprotein; TyG index, triglyceride-glucose index; HOMA-IR, homeostatic model assessment of insulin resistance; Adpn/Lep, adiponectin-leptin index. Data are the median (M) with interquartile range (IQR). Kruskal-Wallis test for the between-group differences. group. The between-group significant differences were caused by the differences between HIIT and VLCHF groups. The most substantial HOMA-IR decrease was in the VLCHF group (median [IQR]: −35.7 [– 20.2; −44.0] %). This decrease in the VLCHF group was caused by changes of both HOMAIR components insulin and glucose. Unlike glucose changes, absolute insulin changes differed by study group (p=0.023, ES 95% CI: small to large) (Table 2 and Figure 2). Absolute changes in the adiponectin/leptin (Adpn/Lep) ratio after 12 weeks differed by study group (p <0.001; ES 95% CI: large) with the differences between the HIIT and Control groups vs. the VLCHF and VLCHF+HIIT groups. The Adpn/Lep ratio increased in the VLCHF group by 120.4 [88.7; 287.1] % and VLCHF+HIIT group by 158.9 [49.5; 540.2] %. These Adpn/Lep increases in the VLCHF and VLCHF+HIIT groups were caused by both the leptin decreases (p<0.001; ES 95% CI: large) and adiponectin increases (p=0.054; ES 95% CI: small to large) (Table 2 and Figure 2). The TyG index and TG/HDL-C ratio significantly decreased in the VLCHF (-0.74 [-2.11; −0.27] and −0.13 [−0.40; −0.04], respectively) and VLCHF+HIIT (−0.68 [−1.89; 0.00] and −0.18 [−0.65; −0.03]) group (Table 2 and Figure 2). Absolute changes in LDL-C after 12 weeks differed by study group (p=0.003; ES 95% CI: small to large) A post hoc analysis revealed differences between the both diet groups (VLCHF, VLCHF+HIIT) and HIIT and Control groups. LDL-C nonsignificantly changed in the VLCHF group by 1.6 [−7.8; 20.0] % and in the VLCHF+HIIT group by 7.4 [−6.1; 20.3] %). However, LDL-C decreased in the HIIT (p=0.012; −8.7 [−12.7; 0.3] %) and Control (p=0.016; −10.1 [−18.3; −1.0] %) groups (Table 2). The complete dataset is reported in the Supplementary Material (Supplementary Tables 3–5). Lp(a), TNF-α, hs-IL−6, IL−1RA, IL−1β, and IL-10 remained mostly under a detection level of the assay. Therefore, no further statistical analyses were conducted (Supplementary Table 6). There were substantial increases in β-hydroxybutyrate concentration (βHB) in the VLCHF and VLCHF+HIIT groups. The highest βHB concentrations were achieved after 2 weeks of VLCHF diet intervention. βHB concentrations in the HIIT and control groups remained within the range between 0.0 to Frontiers in Nutrition | www.frontiersin.org 6May 2022 | Volume 9 | Article 867690 Cipryan et al. VLCHF Diet and HIIT FIGURE 2 | Absolute changes in HOMA-IR, Adpn/Lep ratio, TG/HDL-C ratio, and TyG index after 4 (T1) and 12 (T3) weeks. Frontiers in Nutrition | www.frontiersin.org 7May 2022 | Volume 9 | Article 867690 Cipryan et al. VLCHF Diet and HIIT TABLE 2 | Biochemical variables differences after 12 weeks. HIIT VLCHF VLCHF+HIIT Control Between–group diff. (p–value) 1M(95% CI) 1M(95% CI) 1M(95% CI) 1M(95% CI) Hemoglobin (g/l) −1.5 (−4.5; 1) −1 (−3.5; 0)* −4 (−6; −1.5)* −2 (−4; 1) 0.419 Hematocrit (%) −0.01 (−0.01; 0.00)* 0.00 (−0.01; 0.00) −0.01 (−0.01; 0.00)* −0.01 (−0.01; 0.00) 0.705 Erythrocytes (1012/l) −0.08 (−0.16; 0.02) −0.03 (−0.11; 0.02) −0.12 (−0.20; −0.06)* −0.07 (−0.17; 0.02) 0.343 Thrombocytes (109/l) 2.5 (−9.5; 11.5) −8.0 (−15.0; 6.5) −25.0 (−29.0; −8.0)* 3.0 (−13.5; 15.0) 0.030a Leukocytes (109/l) −0.40 (−0.65; 0.25) 0.00 (−0.65; 0.40) −0.30 (−1.10; 0.15) −0.10 (−0.85; 0.55) 0.760 HbA1c (mmol/mol) 0.0 (−2.0; 2.0) −2.0 (−3.0; 0.0) −2.0 (−4.0; 0.0)* 1.0 (−0.5; 3.0) 0.081 Glucose (mmol/l) −0.02 (−0.38; 0.27) −0.31 (−0.54; −0.05)* 0.01 (−0.50; 0.33) −0.21 (−0.58; −0.04)* 0.316 Triglycerides (mmol/l) 0.03 (−0.33; 0.32) −0.28 (−0.58; −0.10)* −0.20 (−0.70; −0.01)* 0.10 (−0.41; 0.30) 0.161 Cholesterol (mmol/l) −0.46 (−0.56; −0.05)* −0.01 (−0.28; 0.56) 0.29 (−0.22; 0.54) −0.21 (−0.82; 0.01) 0.063 HDL–C (mmol/l) −0.09 (−0.19; 0.03) 0.03 (−0.08; 0.07) 0.07 (−0.05; 0.20) −0.03 (−0.20; 0.04) 0.088 LDL–C (mmol/l) −0.33 (−0.42; −0.08)* 0.06 (−0.12; 0.50) 0.25 (−0.04; 0.50) −0.33 (−0.57; −0.06)* 0.003b Insulin (mU/l) 0.86 (−1.61; 5.27) −2.84 (−3.91; −1.69)** −2.14 (−4.54; 0.37) 0.68 (−2.40; 2.32) 0.023c Leptin (ng/l) −0.19 (−1.36; 0.71) −3.31 (−5.69; −2.59)** −2.40 (−4.42; −1.31)* 4.07 (2.86; 5.53)** <0.001d Adiponectin (mg/l) −1.70 (−8.48; 2.96) 9.30 (1.88; 27.85)* 3.55 (−3.85; 6.41) 3.62 (−43.18; 13.94) 0.054 TG/HDL–C (–) 0.03 (−0.21; 0.29) −0.13 (−0.40; −0.04)* −0.18 (−0.65; −0.03)* 0.04 (−0.16; 0.31) 0.060 TyG index (–) −0.16 (−0.87; 10) −0.74 (−2.11; −0.27)* −0.68 (−1.89; 0.00)* −0.12 (−1.18; 0.56) 0.207 HOMA–IR (–) 0.19 (−0.44; 1.24) −0.75 (−1.13; −0.55)** −0.44 (−1.14; 0.12) −0.01 (−0.73; 0.38) 0.013e Adpn/Lep (–) −0.08 (−1.46; 1.03) 9.34 (6.33; 37.39)** 4.26 (2.24; 13.16)* −3.24 (−21.27; −0.83)* <0.001b Legend: HbA1c, glycated hemoglobin; TG, triglycerides; HDL–C/LDL–C, high/low density lipoprotein; TyG index, triglyceride–glucose index; HOMA–IR, homeostatic model assessment of insulin resistance; Adpn/Lep, adiponectin/leptin ratio. Data are the median differences (1M) between baseline minus 12–week measures with 95% confidence intervals (CI). The complete dataset is reported in the Supplementary Material. Two–tailed Wilcoxon signed–rank test: *significant differences (p<0.05) for baseline vs. 12–week; ** significant differences (p<0.001) for baseline vs. 12–week. Kruskal–Wallis test for the between–group differences. Post–hoc analysis (homogenous subgroups): a– (VLCHF and VLCHF+HIIT), (HIIT, VLCHF and Control); b– (HIIT and Control), (VLCHF and VLCHF+HIIT); c– (VLCHF and VLCHF+HIIT), (HIIT, VLCHF+HIIT and Control); d– Control, HIIT, (VLCHF and VLCHF+HIIT); e– (VLCHF, VLCHF+HIIT and Control), (HIIT, VLCHF+HIIT and Control). 0.3 mmol/l for the whole 12-week intervention. A detailed βHB analysis has already been presented in the parent study (6). Blood Pressure Systolic BP decreased (p <0.05) in all three intervention groups after 8 weeks, when compared to the baseline level, and remained significantly decreased after 12 weeks in the VLCHF+HIIT group. However, systolic BP did not significantly differ by study group at any time point. The between-group differences were shown in diastolic BP after 12 weeks (p=0.049, ES 95% CI: small to medium), with the most pronounced decreases in the VLCHF (1M[95% CI]: −4.0 [−6.8; −0.3] mmHg; ES 95% CI: small to large) and VLCHF+HIIT (−5.3 [−8.0 −3.3] mmHg; ES 95% CI: large) groups (Table 3). DISCUSSION In this randomized controlled trial, we found that VLCHF diet, when compared to HIIT, over 12 weeks in individuals with overfat constitution had substantial benefits for chronic noncommunicable diseases risk factors HOMA-IR, Adpn/Lep ratio and diastolic BP beside the already presented decrease in body mass and VAT (6). However, we did not find significant changes of HOMA-IR from the Control group. Adding HIIT to the VLCHF diet did not caused an extra effect on these variables. We showed that VLCHF diet improved insulin sensitivity and skewed the leptin and adiponectin levels toward anti-inflammatory phenotypes. Despite significant increases in saturated fat intake, we found no significant elevation of LDL-C in the VLCHF and VLCHF+HIIT groups. VLCHF Diet and βHB Less than 50 g/day of CHO was required for the VLCHF groups in this study (29). The aim of the VLCHF diet intervention was not to reduce total energy intake. As such the VLCHF and VLCHF+HIIT groups were encouraged to compensate for the CHO intake restriction by increasing fat intake while maintaining protein consumption. Nevertheless, fat intake was insufficient to keep the total energy intake unchanged (Supplementary Table S2). This is indeed a common situation in real-life conditions. ßHB measures confirmed adherence to the diet in both VLCHF and VLCHF+HIIT groups as has been shown already (13). Notably, total energy intake significantly decreased in the HIIT group despite no diet modification. This effect was likely due to an increased interest in a healthy lifestyle when participating in such a research study. HOMA-IR We found that the HOMA-IR, a frequently used index to evaluate insulin resistance, was substantially reduced after 12 weeks of the VLCHF diet, when compared to HIIT. We showed this improved insulin sensitivity even if the participants with Frontiers in Nutrition | www.frontiersin.org 8May 2022 | Volume 9 | Article 867690 Cipryan et al. VLCHF Diet and HIIT TABLE 3 | Blood pressure outcomes. HIIT VLCHF VLCHF+HIIT Control Between–group diff. (p–value) Systolic PRE 127 (114; 137) 132 (119; 144) 128 (121; 138) 124 (121; 134) – 4 weeks 121 (112; 134) 126 (118; 132)* 125 (115; 134) 124 (117; 131) 0.339 8 weeks 118 (112; 134)* 124 (114; 133)* 120 (114; 134)* 120 (112; 126) 0.990 12 weeks 124 (115; 140) 127 (117; 138) 125 (115; 130)* 120 (118; 133) 0.236 Diastolic PRE 77 (72; 87) 88 (77; 93) 85 (80; 91) 82 (73; 88) – 4 weeks 79 (72; 87) 82 (79; 87) 81 (77; 90)* 79 (74; 86) 0.137 8 weeks 75 (69; 84)* 83 (79; 88) 80 (72; 86)** 77 (71; 83) 0.380 12 weeks 80 (72; 87) 83 (75; 89)* 81 (73; 86)** 79 (73; 82) 0.049a Legend, *different from the baseline (PRE) at p <0.05. Values are shown as median (interquartile range). Two–tailed Wilcoxon signed–rank test, *significant differences (p<0.05) to baseline (PRE); ** significant differences (p<0.001) to baseline (PRE). Kruskal–Wallis test for the between–group differences. Post–hoc analysis (homogenous subgroups): a– (HIIT,VLCHF and Control), (VLCHF, VLCHF+HIIT and Control). overfat constitution were without diabetes and within the normal range of HbA1c. Low carbohydrate diets proved to be more effective than higher carbohydrate (low fat) diets in improving fasting glucose and insulin and insulin sensitivity as measured by HOMA-IR in individuals with obesity and insulin resistance (9) and patients with obesity and non-alcoholic fatty liver disease (10). Not surprisingly, low carbohydrate diets are associated with a large (32 %) increase in remission of diabetes (7). Insulin resistance and excessive body fat are considered among the most important causes of several chronic metabolic and cardiovascular diseases. The cellular and physiological mechanisms are complex and involve adiposity-induced alterations in βcell function, adipose tissue biology, and multi-organ insulin resistance. All these perspectives can be improved with adequate body mass loss (30), which we also showed in this study (6) which may have contributed to the HOMA-IR reduction. Another surrogate measure for the diagnosis of insulin resistance is the TyG index, which is independently and more strongly associated with arterial stiffness in patients with type 2 diabetes than HOMA-IR (31). The TyG index significantly decreased in both the VLCHF group by median 25.9 [IQR: −38.0; 1.2] % and VLCHF+HIIT group by median 23.1 [−54.7; 27.0] %, when the 12-week outcomes were compared to the baseline. However, these favorable changes were not sufficient to prove a significant between-group differences (p=0.207). Nevertheless, we can suggest that a carbohydrate intake restriction might be beneficial not only for the visceral adipose tissue reduction as we showed in the parent study (6), but also for the treatment of the impaired insulin resistance, as already shown elsewhere (29,32,33). The uniqueness of this study, however, lay in including HIIT, alone or in combination with VLCHF diet, into consideration. An exercise intervention program proved to be effective in the treatment of insulin resistance in individuals with overweight/obesity (34), metabolic syndrome (35) or type 2 diabetes, i.e., reduces fasting insulin, HOMA-IR, fasting blood sugar, HbA1c, and body mass index (36). However, we did not show any or additional effect of HIIT on these variables after 12 weeks. This inconsistency can be related to participants characteristics, study duration or other design issues. A diet adjustment seems to be, therefore, crucial within any lifestyle modification for body mass management and reducing health risk variables, despite we still consider physical activity and regular exercise important, e.g., for the maintenance or improvement of the CRF level (6). There is a solid evidence that the CRF level is inversely associated with all-cause, CVD and cancer mortality. A dose-response analyses even showed that a per one-MET increase of the CRF level was associated with 12 %, 13 %, and 7 % reduced risk of all-cause, CVD and cancer mortality (37). Adpn/Lep Ratio We showed significant beneficial changes in the leptin and adiponectin concentrations in the VLCHF group after 12 weeks. The significant decrease in leptin levels also occurred in the VLCHF+HIIT group, whereas the adiponectin increase was not significant. No such beneficial changes were detected in the HIIT and Control groups. A low adiponectin-leptin ratio has been proposed as a promising marker of adipose tissue dysfunction and may lead to chronic systemic inflammation. Leptin is involved in inflammatory responses, and its increased levels are induced by adiposity. In contrast, a decrease in adiposity leads to increased adiponectin levels, which is considered an anti-inflammatory marker (38,39). We have already demonstrated favorable changes in serum adiponectin and leptin concentrations in healthy young individuals following a comparable 12-week VLCHF diet (16). The present study shows that a VLCHF diet can induce similar changes in individuals with overfat constitution. Unlike our previous study with healthy young individuals, in which body mass reduction was only small (16), the decrease in leptin and increase in adiponectin levels in this study might be Frontiers in Nutrition | www.frontiersin.org 9May 2022 | Volume 9 | Article 867690