PLOS ONE

RESEARCH ARTICLE

Efficacy of high-intensity interval training

versus continuous training on serum
myonectin and lipid outcomes in adults with
metabolic syndrome: A post-hoc analysis of a
clinical trial
Jorge L. Petro ID1,2‡, Marı́a Carolina Fragozo-Ramos1‡, Andrés F. Milán1, Juan
a1111111111 C. Aristizabal1, Juan C. Calderón ID1*, Jaime Gallo-Villegas ID3,4*
a1111111111
1 Physiology and Biochemistry Research Group-PHYSIS, Faculty of Medicine, University of Antioquia,
a1111111111
Medellı́n, Colombia, 2 Research Group in Physical Activity, Sports and Health Sciences-GICAFS,
a1111111111 Universidad de Córdoba, Monterı́a, Colombia, 3 Sports Medicine Postgraduate Program and GRINMADE
a1111111111 Research Group, Faculty of Medicine, University of Antioquia, Medellı́n, Colombia, 4 SICOR Center,
Medellı́n, Colombia

‡ JLP and MCFR shared first authorship on this work.

* [email protected] (JCC); [email protected] (JGV)
OPEN ACCESS

Citation: Petro JL, Fragozo-Ramos MC, Milán AF,

Aristizabal JC, Calderón JC, Gallo-Villegas J (2024) Abstract
Efficacy of high-intensity interval training versus
continuous training on serum myonectin and lipid
outcomes in adults with metabolic syndrome: A Background
post-hoc analysis of a clinical trial. PLoS ONE
19(7): e0307256. https://doi.org/10.1371/journal. Myonectin is a myokine with potential effects on the lipid metabolism; however, its regulation
pone.0307256 by exercise in humans remains unclear. We aimed to compare the efficacy of high-intensity
Editor: Aleksandra Klisic, University of interval training low-volume (HIIT) versus moderate-intensity continuous training (MICT) on
Montenegro-Faculty of Medicine, MONTENEGRO serum myonectin, serum lipids, appendicular fat and lean mass, and intramuscular lipids in
Received: January 22, 2024 humans.

Accepted: July 2, 2024

Methods
Published: July 18, 2024
Secondary analysis of a controlled, randomized, clinical trial in adults of both sexes with
Copyright: © 2024 Petro et al. This is an open
access article distributed under the terms of the metabolic syndrome, who underwent a supervised, three-times/week, 12-week treadmill
Creative Commons Attribution License, which program. HIIT (n = 29) consisted of six intervals with one-minute, high-intensity phases at
permits unrestricted use, distribution, and 90% of peak oxygen consumption (VO2peak) for a total of 22 min. MICT (n = 31) trained at
reproduction in any medium, provided the original
60% of VO2peak for 36 min. Serum myonectin was measured using a human enzyme-linked
author and source are credited.
immunosorbent assay. Lipid profile was determined by enzymatic methods and free fatty
Data Availability Statement: We understand the
acids (FFA) were measured by gas chromatography. Fat and lean mass were assessed by
importance of transparency and access to data for
the reproducibility of research, therefore, we have dual-energy X-ray absorptiometry. Intramuscular lipids were measured through proton mag-
submitted a database related to the variables netic resonance spectroscopy.
included in the study as a Supporting information
file (S1 Table).
Results
Funding: The authors received support from the
academic institution as reported in the manuscript. Subjects had a mean age of 50.8±6.0 years and body mass index of 30.6±4.0 kg/m2. Com-
CODI-University of Antioquia (minutes 2020-34909 pared to MICT, HIIT was not superior at increasing serum myonectin (p = 0.661) or linoleic

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PLOS ONE Exercise, myonectin and lipid outcomes in humans

from 22nd February 2021, SIU and minutes 2021- acid (p = 0.263), reducing palmitic (p = 0.286) or stearic acid (p = 0.350), or improving lipid
40430 from 20th October 2021, IIM). The funders profile (all p>0.05), appendicular fat mass index ―AFMI― (p = 0.713) or appendicular lean
did not participate in data collection and analysis,
article writing or submission.
mass percentage ―ALM― (p = 0.810). Compared to baseline, only HIIT significantly
increased myonectin (p = 0.042), with a large effect size, although both interventions
Competing interests: The authors have declared
that no competing interests exist.
reduced AFMI and increased ALM with a large effect size. Lipid profile, FFA and intramuscu-
lar lipids did not change in any intervention group (p>0.05).

Conclusions
Compared to MICT, HIIT low volume did not demonstrate superiority in improving serum lip-
ids. The fact that both training types reduced AFMI without paralleled significant changes in
serum myonectin suggests that this myokine may have a minor effect on short-middle-term
exercise-induced fat mobilization.

Introduction
The metabolic syndrome (MS) is a highly prevalent clinical condition manifested as a combi-
nation of cardiometabolic abnormalities such as hypertension, hyperglycemia, atherogenic
dyslipidemia and abdominal obesity. Low-grade inflammation, endothelial dysfunction,
ectopic fat accumulation (e.g., myosteatosis) and insulin resistance (IR) are key pathophysio-
logic events underlying those clinical abnormalities [1–5].
Aerobic exercise constitutes a central strategy in the prevention and treatment of the MS.
Indeed, different modalities, such as high-intensity interval training (HIIT) and moderate-
intensity continuous exercise (MICT) stimulate beneficial changes in the lipid and FFA pro-
files, and also reduce fat mass (FM) and myosteatosis in patients with overweight, obesity or
MS [6–9]. Both types of exercise further modulate the secretion of myokines, mainly peptides
or proteins expressed and released by muscle fibers which exert either autocrine, paracrine, or
endocrine effects, regulating diverse physiological processes, such as the energy and lipid
metabolism. Myokines could then be primary mediators of the effect of exercise on body com-
position, lipid outcomes and metabolic health [10, 11].
In this context, myonectin has gained attention because it decreases FFA levels in murine
models through the stimulation of their uptake by the adipose tissue and liver [12, 13]. Fur-
thermore, high-fat diet-fed mice exhibited decreased expression levels of myonectin in skeletal
muscle, but aerobic exercise increased its expression, both in slow-twitch and fast-twitch fibers
[12, 14, 15].
The information about the exercise-induced regulation of myonectin in humans, however,
is scant. Most of the evidence coincides in that circulating myonectin levels are lower in sub-
jects with MS and related conditions such as type 2 diabetes mellitus (T2DM) and polycystic
ovary syndrome [16–18]. Then, if myonectin favors the lipid control in humans and is
decreased in the serum of subjects under cardiometabolic disfunction, an exercise intervention
which improves lipid outcomes and cardiometabolic risk factors should increase serum myo-
nectin levels. In fact, a previous report showed that an 8-week MICT intervention in obese
women increased myonectin [19]. That study, however, did not address the effects of the inter-
vention on the lipid profile, body composition or the intramuscular lipids, hindering any con-
clusion on the relevance of the myonectin changes to the aerobic exercise-induced changes in
the lipid profile, body adiposity, and ectopic intramuscular lipid accumulation. Moreover,

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PLOS ONE Exercise, myonectin and lipid outcomes in humans

since HIIT may generate a greater peripheral stimulus on skeletal muscle [20–22], it could
have a greater stimulus on myonectin, something not tested yet. The aim of this study was to
compare the efficacy of a 12-week intervention with HIIT versus MICT on serum myonectin,
serum lipids, appendicular fat and lean mass, and intramuscular lipids in adults with MS.

Materials and methods

Trial design
This is a secondary analysis of a registered, randomized clinical trial (RCT) originally carried
out to evaluate the efficacy of high-intensity interval training low volume (HIIT) or moderate-
intensity continuous training (MICT) on glucose control variables (NCT03087721) [20, 23].
The current reporting of results follows the CONSORT guidelines [24] (S1 Checklist).

Ethics approval
This study was approved by the Research Ethics Committees of the IPS-Universitaria health-
care institution (minutes 159 of 2nd March 2021) and the Faculty of Medicine (minutes 005 of
15th April 2021) at University of Antioquia, in Medellı́n (Colombia), in accordance with the
Resolution number 8430 of 1993 issued by the National Ministry of Health of Colombia, and
the Ethical Principles for Medical Research Involving Human Subjects outlined in the Declara-
tion of Helsinki in 2008. All patients gave written informed consent.

Participants
Each participant underwent a complete medical examination. Socio-demographic information
and personal and family history were also recorded. Blood pressure and heart rate were mea-
sured in the sitting and standing position. For the quantification of physical activity, the vali-
dated Global Physical Activity Questionnaire (GPAQ) was used [25, 26]. Subjects of both sexes
who were 40 to 60 years old and had MS according to the harmonized definition published in
2009, employing specific definitions of waist circumferences standardized for the Colombian
population [27, 28], were included. All participants were recruited from the cardiovascular dis-
ease (CVD) risk program of the IPS-Universitaria and the general population in Medellı́n,
Colombia. People with a vegetarian diet, supplemental consumption (e.g., vitamin D3), inju-
ries or musculoskeletal diseases that prevented exercise; people in a situation of physical, sen-
sory, and cognitive disability; people with a history of cardiopulmonary disease or acute or
chronic inflammatory conditions, cancer, acquired immune deficiency syndrome or T2DM,
and pregnant women were excluded.

Randomization and allocation concealment

The randomization of the participants to either HIIT or MICT groups was performed by the
Clinical trial coordination center, external to the researchers, using the minimization method
with the MinimPy1 v0.3 open software, considering a ratio of 1:1 and the following variables:
age (< 50 and � 50 years), sex (male and female), and body mass index (BMI) (< 30 and � 30
kg/m2), as described in the original protocol (NCT03087721) [20, 23, 29, 30]. This method
does not employ an a priori allocation sequence. Instead, it is an adaptive strategy, which used
the variance as a measure of the distance of the imbalance between groups, and then assigned
the next participant with the "biased coin" method and a base probability of 0.7, in order to
reduce that imbalance. The treatment randomization code for each participant was released to
the researcher in charge of the allocation (JGV) only after all baseline measurements were
completed.

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PLOS ONE Exercise, myonectin and lipid outcomes in humans

Interventions
Supervised treadmill sessions were carried out 3 times per week, for 12 weeks, in the cardiac
rehabilitation and physiotherapy section at the IPS-Universitaria, Medellı́n, Colombia,
between March 2017 and February 2019, always in the mornings. Briefly, all sessions started
with warm-up and finished with cooling-off periods of 3 min at an intensity of 30% of peak
oxygen consumption (VO2peak). HIIT included six intervals of 1 min of high intensity with a
workload of 90% of VO2peak and 2 min with a workload of 50% of VO2peak, for a total duration
of 22 min. MICT trained at 60% of VO2peak for 30 min, for a total duration of 36 min. All
details about the exercise protocols utilized can be found at the Consensus on Exercise Report-
ing Template (CERT) guidelines previously published [20, 23].
VO2peak was chosen to prescribe exercise as it can be considered the best indicator of a per-
son’s cardiorespiratory capacity and physical fitness. The targeted workloads were delivered in
terms of speed and inclination of the treadmill, as previously proposed [31]. VO2peak was
determined in a direct ramp ergospirometry treadmill protocol using an Oxycon Delta by Jae-
ger1 (VIASYS Healthcare GmbH, Germany) at the beginning of the study in previously famil-
iarized subjects, as described [20, 23].

Outcomes
The outcomes assessed in the present post-hoc analysis were: serum myonectin, serum lipid
profile, appendicular FM index (AFMI), appendicular lean mass percentage (ALM), and intra-
muscular lipids. All researchers who analyzed the outcomes were blinded to the groups.
Serum myonectin levels. For myonectin measurements, frozen serum aliquots were
thawed with no extra heat, and 40 μL were poured into the wells of a human myonectin
enzyme-linked immunosorbent assay (ELISA) plate (MyBioSource, MBS1600042, San Diego,
CA, USA). This kit has a detection range of 0.05–100 ng/mL and a sensitivity of 0.03 ng/mL
and showed an intra-assay coefficient of variation (CV) � 4.0% in our laboratory. All plates
were processed by a well-trained investigator blinded to the group coding, according to the
supplier’s instructions. Optical density was determined on a plate reader (Varioskan Lux,
Thermo Scientific, Waltham, MA, USA) at 450 nm.
Serum lipids. Fasting venous blood samples were collected before the intervention and
between 44–48 h after the last session of the intervention program, using serum separator
tubes (BD Vacutainer 367815, USA). The samples were centrifuged at 1300 g for 15 min at
room temperature (Rotofix 32, Hettich, Germany) to obtain the serum in which the lipid pro-
file and FFA were measured.
Low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C) and triglycerides
(TG) were determined by enzymatic analyses using a Dimension1 equipment RXL Max (Sie-
mens, Germany). Typically, the repeatability of these measurements had a CV of � 3%. The
total cholesterol (TC) was calculated using the Friedewald formula [32].
The extraction of FFA was performed by methylation following the Folch method [33]. For
this, 40 μL of 50 mg/mL tridecanoic acid and 2 mL of chloroform/methanol (2:1) were added
to 100 μL of serum. The mixture was shaken for 1 min and 1 mL of saturated sodium chloride
was added. Then, it was centrifuged at 3400 rpm for 7 min and the organic phase was separated
and dried in a bath at 90˚C. The dry extract was dissolved in 1 mL of hexane and deposited on
aminopropyl columns (200 mg). The separation of the FFA was through gas chromatography
[34], using a standard (FAME Mix of 37 components: C4-C24, Supelco1, USA). An Agilent
chromatograph (7890B, USA) with flame ionization detector and TR-CN100 capillary column
(60 m x 250 μm x 0.20 μm ID) was used. Helium was used as carrier gas. We analyzed the pal-
mitic 16:0, stearic 18:0 and linoleic 18:2n-6 FFA. Although we also measured arachidonic acid

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PLOS ONE Exercise, myonectin and lipid outcomes in humans

(C20:4n6), nervonic acid (C24:1n9), 11-eicosenoic acid (C20:1n-9c) and adrenic acid (C22:4n-
6c), they could not be quantified for all participants, making the number of values available for
statistical analysis low. The CV of repeated measurements was � 1%.
Fat and lean mass. Participants were weighed on an electronic scale with 0.1 kg accuracy
(Omron1 HBF-510LA, Omron Healthcare, Inc., Illinois, United States), while wearing mini-
mal clothes. Height was measured to the nearest 0.1 cm with a stadiometer (Seca, Hamburg,
Germany). Waist circumference was measured with a fiberglass anthropometric tape at the
intermediate point between the lower edge of the last rib and the iliac crest, in the horizontal
plane. The BMI was calculated as body mass (kg)/height (m2).
Body composition was assessed using Dual-energy X-ray absorptiometry (DXA) with a Dis-
covery Wi DXA system1 (Hologic, USA) and the Hologic APEX v4.5.3 software (Hologic,
USA), as this technique has shown excellent accuracy in reporting both fat and lean mass [35].
The subjects were examined in the morning under fasting conditions, but well hydrated status,
as indicated by a measured urinary density between 1000 and 1025. For this study, AFMI
([arms FM (kg) + legs FM (kg)]/height (m)2) and ALM ([appendicular lean mass (kg)/total
body mass (kg)] x 100) were considered relevant, because they are associated with an elevated
risk of cardiometabolic diseases, disability, and mortality [36, 37]. These appendicular mea-
surements complement our recent report in the same population comparing the effect of HIIT
vs MICT on depots of global (total fat mass, fat percentage) and central (android, visceral,
gynoid) fat [8].
Intramuscular lipids content. The intramuscular lipid content was non-invasively mea-
sured through proton magnetic resonance spectroscopy (1H-MRS), following international
recommendations and protocols previously published [17, 20, 38]. Briefly, for the acquisition,
a voxel of 15 x 15 x 35 mm3 was placed inside the right vastus lateralis muscle (VLM), due to
its relevance to the development of metabolic diseases in humans [39, 40], and the spectra
were collected using a flexible coil in a 3T Magnetom Skyra system (Siemens Healthcare,
Erlangen, Germany). The spectra were processed with the jMRUI v5.2 free software (http://
www.jmrui.eu) [41] to adjust the water reference to 4.7 parts per million (ppm), apodize and
subtract peaks from metabolites other than methylenes (CH2) from the intramyocellular
(IMCL, 1.3 ppm) and the extramyocellular (EMCL, 1.5 ppm). Then, the integrals of the CH2
peaks of IMCL and EMCL were quantified. Water amplitude was analyzed in spectra obtained
without water suppression. The relaxation times (T1 and T2) of IMCL-CH2 and EMCL-CH2
were taken from Valaparla et al. [42] and the absolute concentration of intramuscular lipids
per kilograms of wet weight (mmol�kg−1 ww) was estimated using the validated equation of
Szczepaniak et al. [43, 44]. In our hands, the reliability of the quantification of intramuscular
lipids using 1H-MRS has a high intraclass correlation coefficient (ICC, 0.98, ―0.97–0.99―).

Statistical methods
Results are reported as mean ± SD for normally distributed datasets and as median and inter-
quartile range (IQR) for skewed ones. The normality of the data and the homogeneity of vari-
ances were assessed using the Shapiro-Wilk test and Levene’s test, respectively. Comparisons
at baseline were performed with the unpaired Student’s t-test or Mann-Whitney U test for
quantitative variables, and Fisher’s exact test for categorical variables.
Intention-to-treat (HIIT, n = 29; MCT, n = 31) analyses were performed. Paired t-test and
estimation statistics [45] were used to test the means change within each intervention (pre vs
post) and then analysis of covariance (ANCOVA) was used to estimate the effect of HIIT com-
pared to MICT (between interventions) on the outcomes included in Table 2. Because
ANCOVA has greater statistical power compared to classical methods such as analysis of

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PLOS ONE Exercise, myonectin and lipid outcomes in humans

variance and change-score analysis, it is currently considered the optimum statistical method
for the analysis of continuous outcomes in RCT [46]. The results are presented as the adjusted
mean difference (95% confidence interval ―CI―). A re-randomization test was performed
using permutations for inference purposes. The use of the re-randomization method with per-
mutations is justified by its ability to provide valid statistical inferences and control the fre-
quency of type I errors in situations where conventional methods may not be adequate, such
as in clinical trials with small sample sizes where minimization was used, thereby ensuring the
integrity and reliability of the results [29, 47, 48].
Effect sizes were estimated with Cohen’s d for repeated measures, using the correction for
the correlation of the pre-post values [49]. Myonectin results are shown as natural logarithm
(Ln) because of its not normal distribution.
Given that the subsample for the intramuscular lipid measurement was lower (HIIT, n = 9;
MICT, n = 12) than the other outcomes, only intragroup comparisons (pre vs post) were per-
formed, so they were not included in Table 2. These comparisons were carried out with Wil-
coxon test instead of t-test and these analyses can only be considered exploratory.
In this study, the level of statistical significance was set at p<0.05 for all tests. Data analyses
were performed using STATA1 v14.0 (StataCorp LLC, USA) and SPSS1 Statistics v21.0
(IBM, USA).

Results
Study participants
Three hundred seven people were contacted for eligibility. One hundred thirty-eight were
cited for clinical evaluation, and 60 fulfilled the criteria for participation and were randomly
assigned to either the HIIT (n = 29) or MICT (n = 31) groups. One participant was lost to fol-
low-up and one subject was excluded after being diagnosed with T2DM one month after start-
ing participation in the project (Fig 1).

Baseline characteristics
The mean age of the participants was 50.8 ± 6.0 years, BMI was 30.6 ± 4.0 kg/m2 and their
median body weight was 75.5 (67.5–84.0) kg. Most participants were female (70%). As
expected, the participants were mostly overweight or obese, with dyslipidemia and other CVD
risk factors (Table 1, S1 Table). Baseline characteristics were similar between the two groups,
except for the use of statins (Table 1).

Outcomes
Serum myonectin and serum lipids. Compared to baseline values, changes in circulating
levels of serum myonectin and LDL-C, HDL-C, TG, TC, TC/HDL and the evaluated FFA were
not statistically significant in either group (Table 2). However, HIIT showed a trend to increase
after the intervention when compared with a paired t-test. Interestingly, the more robust estima-
tion statistics approach revealed a significant change, with a p = 0.042 (Fig 2). Also, Fig 3 con-
firmed an increase in myonectin with a large effect size only in the HIIT group, with marginal
changes for the other serum lipids. Finally, there were no significant differences in myonectin
and other circulating lipids between the training groups after the intervention (Table 2, Fig 4).
Fat and lean mass. MICT significantly decreased AFMI and both HIIT and MICT
reduced it with a large effect size (HIIT, 0.09 kg [95% CI 0.03 to 0.20] Cohen’s d: -2.22 and
MICT, 0.10 kg [95% CI 0.02 to 0.22] Cohen’s d: -2.41). Similarly, MICT increased ALM signif-
icatively, and both interventions increased it with a large effect size (HIIT, 0.28% [95% CI 0.04

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PLOS ONE Exercise, myonectin and lipid outcomes in humans

Fig 1. Flow diagram of the clinical trial. HIIT, high-intensity, low-volume interval training; MICT, moderate
intensity continuous aerobic training.
https://doi.org/10.1371/journal.pone.0307256.g001

to 0.61] Cohen’s d: 1.43 and MICT, 0.37% [95% CI 0.02 to 0.72] Cohen’s d: 1.60) compared to
the baseline (Table 2, Fig 3). There were no differences in AFMI and ALM changes between
HIIT and MICT groups after the interventions (Table 2, Fig 4).
Intramuscular lipids content. In HIIT, compared to baseline values, IMCL (pre: 12.50
mmol�kg−1 ww (4.80–35.12) vs post: 6.96 mmol�kg−1 ww (4.99–22.80) (p = 0.893)), EMCL

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PLOS ONE Exercise, myonectin and lipid outcomes in humans

Table 1. Demographics and clinical characteristics of the subjects of the study.

Characteristics HIIT (n = 29) MICT (n = 31) p*
Demographics
Age (years) mean (SD) 51.5 ± 6.0 50.2 ± 6.0 0.409
Female sex, n (%) 21 (72.4) 21 (67.7) 0.782
Worker, n (%) 16 (55.2) 21 (67.7) 0.408
Unemployed, n (%) 12 (41.3) 10 (32.25)
Retired, n (%) 1 (3.4) 0 (0.0)
Clinics
Hypertension, a n (%) 10 (34.5) 12 (38.7) 0.312
Dyslipidemia, b n (%) 25 (86.2) 25 (80.6) 0.732
Overweight or obese, c n (%) 27 (93.1) 29 (93.5) 1.000
Impaired fasting glycemia, d n (%) 11 (37.9) 16 (51.6) 0.312
Anthropometrics
BMI (kg/m2), mean (SD) 30.4 ± 4.0 30.8 ± 4.1 0.731
Waist circumference (cm), median (IQR) 94.0 (89.1–103.2) 94.5 (90.4–102.6) 0.544
Total fat mass (kg), mean (SD) 29.1 ± 6.1 29.7 ± 8.3 0.758
Total lean mass (kg), median (IQR) 40.3 (36.0–51.7) 43.7 (38.8–51.7) 0.318
Habits
Caloric intake, mean (SD) 2117.2 ± 604.8 2118.0 ± 794.6 0.997
Physical activity � 600 MET, n (%) 19 (65.5) 15 (48.4) 0.203
Current smoker, n (%) 1 (3.4) 3 (9.7) 0.613
Biochemical
Fasting insulin (mUI/L), median (IQR) 14.7 (12.2–18.8) 14.3 (11.2–19.4) 0.663
Fasting glycemia (mg/dL), mean (SD) 98.1 ± 8.8 99.8 ± 8.7 0.454
HbA1c (%), mean (SD) 5.63 ± 0.28 5.72 ± 0.34 0.311
Medications
ACEI or ARA II, n (%) 9 (31.0) 11 (35.5) 0.788
Beta-blockers, n (%) 4 (13.8) 2 (6.5) 0.417
Calcium antagonists, n (%) 2 (6.9) 0 (0.0) 0.229
Diuretics, n (%) 5 (17.2) 6 (19.4) 1.000
Aspirin, n (%) 2 (6.9) 0 (0.0) 0.229
Statins, n (%) 8 (27.6) 2 (6.5) 0.039
Metformin, n (%) 2 (6.9) 0 (0.0) 0.229

Values are n (%), mean (SD) or median (IQR). SD, standard deviation; IQR, interquartile range; HIIT, high-intensity, low-volume interval training; MICT, moderate-
intensity continuous aerobic training; BMI, Body mass index; BP, blood pressure; HbA1c, glycated hemoglobin; ACEI, Angiotensin-converting enzyme inhibitors;
ARAII; Angiotensin II receptor antagonists;
a
BP � 140/90 mmHg or treatment with antihypertensive drugs;
b
One or more of the following criteria: LDL cholesterol �130 mg/dL, triglycerides �150 mg/dL or previous diagnosis of dyslipidemia;
c
BMI � 25 kg/m2;
d
fasting blood glucose � 100 mg/dL and < 126 mg/dL;
*p-value of Student’s t-test or Mann-Whitney U-test for quantitative variables and Fisher’s exact test for categorical variables between two groups.

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(pre: 35.50 mmol�kg−1 ww (28.95–56.12) vs post: 35.79 mmol�kg−1 ww (25.89–77.95)

(p = 0.401)) and total intramuscular lipid content (pre: 40.70 mmol�kg−1 ww (35.90–48.90) vs
post: 38.77 mmol�kg−1 ww (28.92–58.55) (p = 0.715)) remained unaltered. Similarly, no
changes were observed in MICT compared to baseline values in IMCL (pre: 9.15 mmol�kg−1
ww (5.50–11.33) vs post: 8.51 mmol�kg−1 ww (6.50–9.96) (p = 0.401), EMCL (pre: 25.85

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PLOS ONE Exercise, myonectin and lipid outcomes in humans

Table 2. Myonectin, serum lipids profile, free fatty acids, fat mass and lean mass results at baseline and after 12 weeks of intervention in the two training groups.
Intention-to-treat analysis.
Outcomes HIIT (n = 29) a p MICT (n = 31) a p HIITc MICTc Absolute 95% CI p
Before After valueb Before After valueb Adjusted Adjusted effect of valued
mean mean HIITd
Mean SD Mean SD Mean SD Mean SD Lower Upper
Myonectin Ln 0.23 0.68 0.29 0.72 0.055 0.14 0.51 0.24 0.70 0.340 0.24 0.29 0.05 -0.17 0.27 0.661
Serum lipids
profile
LDL-C 149.53 44.57 149.76 49.02 0.970 145.93 59.18 144.80 42.17 0.807 148.18 146.37 -1.80 -16.10 12.48 0.800
(mg/dL)
HDL-C 46.37 11.20 47.22 14.22 0.664 43.61 9.43 43.59 10.18 0.985 46.03 44.78 -1.25 -6.12 3.62 0.609
(mg/dL)
TG (mg/ 205.57 91.27 190.45 98.54 0.262 178.84 104.32 177.07 106.25 0.908 181.71 185.81 4.10 -33.76 41.96 0.829
dL)
TC (mg/ 237.06 56.57 234.85 60.52 0.749 225.96 60.65 224.49 44.59 0.819 230.96 228.38 -2.58 -19.59 14.43 0.762
dL)
TC/HDL 5.26 1.14 5.16 1.15 0.622 5.31 1.46 5.37 1.43 0.740 5.18 5.35 0.17 -0.33 0.68 0.492
Free fatty
acids (FFA)
Palmitic 0.19 0.11 0.17 0.09 0.502 0.19 0.10 0.20 0.10 0.699 0.17 0.20 0.02 -0.02 0.07 0.286
acid (μg/dL)
Stearic acid 0.08 0.03 0.08 0.04 0.745 0.08 0.05 0.09 0.05 0.449 0.01 0.01 0.01 -0.01 0.03 0.350
(μg/dL)
Linoleic 0.13 0.07 0.11 0.05 0.188 0.15 0.13 0.15 0.13 0.810 0.11 0.14 0.03 -0.25 0.09 0.263
acid (μg/dL)
Fat mass and
lean mass
AFMI (kg/ 5.30 1.58 5.20 1.59 0.111 5.49 1.87 5.36 1.84 0.015 5.30 5.27 -0.02 -0.18 0.12 0.713
m2)
ALM (%) 23.43 3.50 24.72 3.43 0.082 24.41 4.03 24.79 3.97 0.041 24.72 24.78 0.05 -0.40 0.52 0.810
a
Unadjusted means and SD,
b
paired t-test for the change after each intervention,
c
means adjusted for age, sex, body mass index and baseline value using ANCOVA,
d
difference between HIIT and MICT adjusted means, and ANCOVA P value for the difference between HIIT and MICT adjusted means.
SD, standard deviation; HIIT, high-intensity, low-volume interval training; MICT, moderate intensity continuous aerobic training; CI, confidence interval; ANCOVA,
analysis of covariance; LDL-C, low-density lipoprotein; HDL-C, high-density lipoprotein; TG, triglycerides; TC, total cholesterol; AFMI, appendicular fat mass index;
ALM, appendicular lean mass percentage.

https://doi.org/10.1371/journal.pone.0307256.t002

mmol�kg−1 ww (19.18–34.45) vs post: 20.86 mmol�kg−1 ww (14.83–42.95) (p = 0.929)) and

total intramuscular lipid content (pre: 30.06 mmol�kg−1 ww (23.28–42.24) vs post: 25.58
mmol�kg−1 ww (20.77–40.91) (p = 0.735)). No differences were found between groups at the
end of the intervention (p = 304).

Discussion
The main findings of this study were: (i) HIIT was not superior to MICT in inducing changes
in myonectin values or in improving serum lipid profile. Myonectin significantly increased,
with a large effect size, only in the HIIT group; (ii) HIIT was not superior to MICT in changing
AFMI and ALM, although both interventions improved body composition; (iii) the interven-
tions did not decrease intramuscular lipids.

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PLOS ONE Exercise, myonectin and lipid outcomes in humans

Fig 2. Comparison of the effect of the intervention on serum myonectin. Cumming estimation plot for the paired
mean difference of the effect of HIIT or MICT on myonectin. The raw data is plotted on the upper axes. Each paired
set of observations (pre and post) is connected by a black line for each intervention type. The lower axes show each
paired mean difference (black dots) plotted as a bootstrap sampling distribution (light gray). The 95% CI are depicted
as vertical error bars. Paired means difference for HIIT was 0.06 [95% CI 0.01, 0.12], p = 0.042. Paired means
difference for MICT was 0.09 [-0.07, 0.34], p = 0.366. HIIT, high-intensity, low-volume interval training; MICT,
moderate intensity continuous aerobic training; 95% CI, confidence intervals.
https://doi.org/10.1371/journal.pone.0307256.g002

The effect of HIIT and MICT on serum myonectin levels

The skeletal muscle is an endocrine organ with a prominent role in the metabolic control and
the pathophysiology of cardiometabolic diseases [11]. Myonectin has gained special attention
for its potential to mediate the interaction between muscle, exercise, and lipid metabolism
[12, 13]. However, the literature regarding this interaction is inconsistent.
One study assigned 80 healthy or overweight/obese women to exercise (n = 34) and con-
trol (n = 46) groups. The exercise program comprised three weekly 45-minute sessions of
MICT for 8 weeks that included running with 50–70% of maximum heart rate. The authors
showed that the serum myonectin level increased significantly in the experimental group
[19]. On the other hand, Bahremand and coworkers did not find myonectin changes in a

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PLOS ONE Exercise, myonectin and lipid outcomes in humans

Fig 3. Effect size of the comparison between baseline and after intervention on outcomes in the intention-to-treat
analysis (HIIT n = 29, MICT n = 31). Effect size (95% CI) of the comparison between baseline and after 12 weeks of HIIT or
MICT intervention on myonectin, serum lipids profile, free fatty acids, fat mass and lean mass. HIIT, high-intensity, low-
volume interval training; MICT, moderate intensity continuous aerobic training; LDL-C, low-density lipoprotein; HDL-C,
high-density lipoprotein; TG, triglycerides; TC, total cholesterol; TC/HDL, total cholesterol to HDL-C ratio; AFMI,
appendicular fat mass index; ALM, appendicular lean mass percentage; 95% CI, confidence intervals.
https://doi.org/10.1371/journal.pone.0307256.g003

study performed in 30 healthy women that were randomly assigned to CrossFit (n = 16) and
concurrent aerobic plus resistance training (CT; n = 14) groups, exercising three sessions per
week for 8 weeks [50].
Here, we only found relevant increases in myonectin for the HIIT group, thus confirming
an exercise-induced increase in myonectin, but only under some conditions. Interestingly, the
work of Bahremand and ours (see the report of results of the primary trial [20]) were effective
at modifying cardiovascular (VO2max, heart rate at rest), metabolic (FM, glycaemia, homeo-
static models of insulin resistance) and muscular variables (lean mass, power output, strength),
showing that the exercise interventions were well delivered [20, 50].
These apparently contrasting findings may have different explanations: i) myonectin
changes may not be necessary for changes in fat depots but may be necessary for changes in
serum lipids, in agreement with findings suggesting that clear changes in the lipid profile in
humans can be observed after 16 weeks of training [51], ii) the non-significant changes, even
the small effect size increase observed for myonectin in MICT, may reflect early changes

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PLOS ONE Exercise, myonectin and lipid outcomes in humans

Fig 4. Effect size of the intervention on outcomes in the intention-to-treat analysis (HIIT n = 29, MICT n = 31).
Effect size (95% CI) of the comparison between the two training groups on myonectin, serum lipids profile, free fatty
acids, fat mass and lean mass after 12 weeks of intervention. HIIT, high-intensity, low-volume interval training; MICT,
moderate intensity continuous aerobic training; LDL-C, low-density lipoprotein; HDL-C, high-density lipoprotein;
TG, triglycerides; TC, total cholesterol; TC/HDL, total cholesterol to HDL-C ratio; AFMI, appendicular fat mass index;
ALM, appendicular lean mass percentage; 95% CI, confidence intervals.
https://doi.org/10.1371/journal.pone.0307256.g004

sufficient to mobilize lipids from fat depots, which, if augmented or maintained for a long
time, may afterwards induce effective changes in serum lipid variables, iii) exercise-induced
changes in myonectin are slow, of low magnitude, dependent on the type of intervention and
with an effect on other lipid variables on the long term. The last hypothesis seems to be the
most supported by the evidence. First, we have shown that lower myonectin levels are associ-
ated with an increased android/gynoid ratio [17], and in this work and in a recently pub-
lished article, with the same population, we saw that several global, central and appendicular
FM depots are effectively reduced after a 12-weeks intervention with aerobic exercise [8].
Noticeably, MICT was even superior in reducing android fat mass compared to HIIT [8],
but here, the most important change in myonectin was in the HIIT group, likely indicating
that myonectin is not a primary mediator of the changes in these fat depots. The larger
change in myonectin found in HIIT can be explained because of the larger muscle oxidative
metabolic activation, including more activation of fibers type II, under this type of exercise
compared with MICT [20, 21]. Since myonectin is produced by both fibers type I and II [12],
it is possible that a larger strain imposed during the intervention on fibers type II, and not

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PLOS ONE Exercise, myonectin and lipid outcomes in humans

only on fibers type I, increases the amount of muscle mass releasing myonectin. Instead, the
effect of MICT on myonectin seems to be delayed and of a lower magnitude. Second, myo-
nectin levels do not correlate with FFA in subjects with MS under resting conditions [17],
therefore, myonectin changes may not be as relevant as initially thought to induce changes
in serum lipids. Third, acute, short term physical exertions, or lipid infusions, do not change
myonectin in blood [52, 53]. Fourth, the values of circulating myonectin generally found in
humans (~0.1–400 ng/mL) [17–19] are up to 4 orders of magnitude lower than those shown
to influence lipid metabolism and other functions in different murine and cellular models
(~1–5 μg/mL) [12, 54, 55], indicating that in several papers the authors have used pharmaco-
logical concentrations of myonectin rather than physiological concentrations to accelerate
and potentiate any biological effect.

The effect of HIIT and MICT on serum lipid profile

Studies comparing the effect of HIIT versus MICT on serum lipids in patients with MS, obe-
sity, and T2DM have shown contradictory results. The positive effects of HIIT alone on LDL
have been previously described for Da Silva et al. in a study that enrolled thirty-nine subjects
with MS to a 12-weeks exercise intervention [56]. Another study, however, did not show any
effect of HIIT on LDL and TC in obese men, even when applied for 16 weeks. Interestingly,
the HIIT-MICT alternation rendered better results, improving LDL and TC [51]. In the same
sense, Tjønna et al. compared HIIT versus MICT in thirty-two patients with MS and did not
find differences between both types of training on LDL, TG and TC [57]. Similarly, other RCT
and recent meta-analysis noticed that HIIT was not superior to MICT for modifying LDL, TG,
TC or TC/HDL [58, 59]. Together, HIIT is not superior to MICT in improving the lipid profile
in subjects with cardiometabolic diseases.
The minor differences found between the studies may be due to the components of the
HIIT used (intensity, duration and number of intervals, recovery periods or volume) and dura-
tion of the intervention. In addition, is necessary to take in account the time of day of exercise
training (morning vs evening). A recently published study, including 25 overweight or obese
participants that were allocated to HIIT in the morning (06:30 hours), in the evening (18:30
hours) or no exercise, observed that fasting blood glucose, insulin, TG, TC and LDL-C concen-
trations decreased only in participants allocated to evening exercise training, as opposed to
our protocol, suggesting that the time of day of training would modulate the effect of the exer-
cise on serum metabolomics [60]. In our case, all exercise sessions were performed in the
morning, likely reducing their effectiveness.
Studies evaluating the effectiveness of HIIT on FFA compared to MICT in the context of
MS suggest that subjects benefit from high volume (higher frequency, longer duration,
cointerventions), instead of low volume. For instance, a study conducted in nineteen class
II and III obese subjects that were assigned to two weeks of continuous training at the
intensity eliciting the maximal fat oxidation (Fatmax) versus HIIT, showed a decrease of
FFA in the group allocated to Fatmax (P<0.002) [61]. Also, a decrease in saturated fatty acid
(SFA) and n-6 polyunsaturated fatty acid (PUFA) levels with parallel increase of monoun-
saturated fatty acid (MUFA) and n-3 PUFA was observed after a multidisciplinary inter-
vention that included a calorie-restricted diet (10–40%) and increased continuous physical
activity (at least 60 min/5 days a week) during six months [62]. Thus, our intervention
seemed to be of lower volume than that required to elicit measurable changes in FFA, some-
thing which should be considered when prescribing HIIT modalities to subjects with lipid
alterations.

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PLOS ONE Exercise, myonectin and lipid outcomes in humans

The effect of HIIT and MICT on body composition and intramuscular

lipids content
We recently showed that both MICT and HIIT comparably reduced total FM and gynoid FM,
and increased total lean mass in adults with MS, while MICT had additional benefits by reduc-
ing the android and the android/gynoid FM ratio [8]. The rather similar beneficial effects of
HIIT and MICT, or in any case the no superiority of HIIT, on body composition has been
acknowledged in other studies as well [63–65]. In the present study, we extended the analysis
to AFMI and ALM and showed the significant beneficial effect of MICT compared to HIIT on
AFMI, but HIIT and MICT showed a trend or a significant gain in ALM. This variable has
been proposed as a more reliable parameter for the definition of sarcopenic obesity permitting
an adequate characterization of the obesity phenotype [66]. Anyhow, both interventions
improved body composition with a large effect size. These results highlight that the interven-
tion was well delivered. Moreover, the reduction of AFMI in MICT did not require the
increase of myonectin, reinforcing the hypothesis that this myokine may have a minor effect
on exercise-induced fat mobilization in humans in the short-middle term.
Although skeletal muscle tissue typically contains small amounts of adipose tissue, several
CVD risk factors can lead to an excess of intramuscular deposition of adiposity, in a phenome-
non called myosteatosis [67]. Although aerobic exercise of over 10 weeks improves muscle
quality in adult populations at risk of developing obesity and sarcopenia-related disability [9],
our study failed to show changes in intramuscular lipids content after either HIIT or MICT
interventions. Even when our sample size for this outcome was small, we have seen that myo-
nectin does not correlate with intramuscular lipids in a larger sample of subjects with MS [17].
In agreement with the above discussion, we speculate that myonectin may have a minor role
in mobilizing intramuscular lipids, however, a larger and longer study should address this spe-
cific aim in the future.

Strengths, limitations, and final remarks

This is one of the few studies comparing the effects of HIIT and MICT on serum myonectin
levels and lipid outcomes in people with MS, carrying out the evaluation of body composition
and FFA by DXA and gas chromatography respectively, methods with excellent accuracy. Fur-
thermore, we performed robust measures of FFA clinically relevant, closely correlated with
metabolic status and the prediction of development of MS [68, 69]. Also, both training groups
improved VO2peak around the same magnitude as previously reported [20, 70], indicating a
good delivery of the intervention. A post-hoc analysis shows a power of 0.9 to detect a pre vs
post change in myonectin in the HIIT group, and of 0.7 in the MICT group. The power to
detect intergroup differences (HIIT vs MICT) in the ANCOVA analysis at the end of the inter-
vention, after multiple adjustments, is 0.75. Although the results presented here were con-
firmed with a model of re-randomization with permutations (not shown), supporting the
validity of the ANCOVA analyses, we cannot fully rule out an error type II when testing the
effect of MICT on myonectin. A future work with a larger sample and follow up for over 12
weeks may confirm the hypothesis according to which myonectin responds lower and slower
to MICT interventions. Although some participants were on statins, we have shown that they
do not change any relationship between myonectin and metabolic outcomes [17], so they are
not expected to affect our results. Finally, both interventions were safe as we recently reported
[71], helping ensure a low rate of losses.
Although this is the first study in humans evaluating the relationship between myonectin
and myosteatosis analyzed by 1H-MRS after an exercise intervention, the scope of the results
was limited by the restricted number of subjects and the small changes observed, reducing the

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PLOS ONE Exercise, myonectin and lipid outcomes in humans

statistical power for finding differences between the groups. The number of subjects was cho-
sen as a random subsample due to the high costs and demanding sources of the technique, ren-
dering these results thus exploratory, and reinforcing the idea that future works in the field
should include a sizeable sample. Ultimately, the results presented here need to be confirmed
in further research given the characteristics of the post-hoc analysis.

Conclusions
Compared to MICT, HIIT was not superior at increasing myonectin or improving serum lipid
markers. The fact that both training types for 12 weeks reduce fat depots without preceding
parallel changes in serum myonectin suggests that this myokine may have a minor effect on
exercise-induced fat mobilization in the short-middle term. Long-term MICT stimulation
may be necessary to elicit sizeable increases in serum myonectin.

Supporting information
S1 Checklist. CONSORT checklist.
(DOCX)
S1 Table Dataset. Contains a dataset of the variables included in this manuscript.
(XLSX)

Acknowledgments
The authors thank the Faculty of Medicine, Universidad de Antioquia, Laboratorio Docente
Asistencial, IPS-Universitaria, Indeportes Antioquia, Pablo Tobón Uribe Hospital and SICOR
Center, all in Medellı́n, Colombia. We also thank Professor Norman Balcázar at University of
Antioquia for help with the ELISA plate reader.

Author Contributions
Conceptualization: Jorge L. Petro, Juan C. Calderón, Jaime Gallo-Villegas.
Investigation: Marı́a Carolina Fragozo-Ramos, Juan C. Calderón, Jaime Gallo-Villegas.
Methodology: Jorge L. Petro, Marı́a Carolina Fragozo-Ramos, Andrés F. Milán, Juan C. Aristi-
zabal, Juan C. Calderón, Jaime Gallo-Villegas.
Project administration: Juan C. Calderón, Jaime Gallo-Villegas.
Supervision: Juan C. Calderón, Jaime Gallo-Villegas.
Writing – original draft: Jorge L. Petro, Marı́a Carolina Fragozo-Ramos, Juan C. Calderón,
Jaime Gallo-Villegas.
Writing – review & editing: Jorge L. Petro, Marı́a Carolina Fragozo-Ramos, Juan C. Aristiza-
bal, Juan C. Calderón, Jaime Gallo-Villegas.

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