Eur J Appl Physiol

DOI 10.1007/s00421-017-3535-y

ORIGINAL ARTICLE

Change in maximal fat oxidation in response to different regimes

of periodized high-intensity interval training (HIIT)
Todd A. Astorino1 · Ross M. Edmunds2 · Amy Clark1 · Rachael Gallant1 ·
Leesa King1 · Gina M. Ordille1 · Brendyn Heath1 · Matthew Montell1 ·
Jason Bandong1 

Received: 18 July 2016 / Accepted: 3 January 2017

© Springer-Verlag Berlin Heidelberg 2017

Abstract  Conclusions  Our results refute the widely reported

Purpose  Increased capacity for fat oxidation (FatOx) is increases in capacity for FatOx demonstrated with HIIT,
demonstrated in response to chronic endurance training as which is likely due to marked day-to-day variability in
well as high-intensity interval training (HIIT). This study determinations of MFO and exercise fat oxidation as well
examined changes in maximal fat oxidation (MFO) in as the heterogeneity of our sample.
response to 20 sessions of periodized HIIT in an attempt
to identify if various regimes of HIIT similarly augment Keywords  Interval training · Fat utilization · Cycle
capacity for FatOx. ergometry · Carbohydrate oxidation · MFO
Methods  Thirty-nine habitually active men and women
(mean age and V ­ O2max = 22.5 ± 4.4  year and 40.0 ± 5.6 Abbreviations
mL/kg/min) completed training and 32 men and women ANOVA Analysis of variance
with similar physical activity and fitness level served as CHO Carbohydrate
non-exercising controls (CON). Training consisted of ten CHOOx Carbohydrate oxidation
sessions of progressive low-volume HIIT on the cycle Fatmin Minimum fat oxidation
ergometer after which participants completed an additional FatOx Fat oxidation
ten sessions of sprint interval training (SIT), high-volume FFM Fat free mass
HIIT, or periodized HIIT, whose assignment was rand- HIIT High-intensity interval training
omized. Before and throughout training, MFO, FatOx, and HRmax Maximal heart rate
carbohydrate oxidation (CHOOx) were assessed during MFO Maximal fat oxidation
progressive cycling to exhaustion. PPO Peak power output
Results  Compared to CON, there was no effect of HIIT RER Respiratory exchange ratio
on MFO (p = 0.11). Small increases (p = 0.03) in FatOx SIT Sprint interval training
were evident in response to HIIT leading to an additional VO2max Maximal oxygen uptake
4.3 g of fat oxidized, although this value may not be clini-
cally meaningful.
Introduction

Communicated by William J. Kraemer. Over 30  year ago, Holloszy and Coyle (1984) identified
that significant increases in fat oxidation (FatOx) and
* Todd A. Astorino reductions in carbohydrate oxidation (CHOOx) occur
astorino@csusm.edu
with chronic endurance training that mediate improved
1
Department of Kinesiology, CSU—San Marcos, 333. S. performance. Subsequently, Hurley et  al. (1986) demon-
Twin Oaks Valley Road, UNIV 320, San Marcos, CA, USA strated that 12 weeks of strenuous endurance exercise in
2
Department of Physical Therapy, SUNY—Stony Brook, men led to significant increases in FatOx and concomitant
Stony Brook, NY, USA sparing of muscle glycogen, with the former explained

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by greater use of muscle triglyceride. In cyclists, Rom-
ijn et  al. (1993) reported that FatOx peaked at a mod-
erate intensity equal to 65% maximal oxygen uptake
(%VO2max) after which it declined at 85%VO2max, a
finding supported by another investigation (Van Loon
et  al. 2001). This interest in identifying peak rate of
FatOx was furthered by a cross-sectional study (Venables
et  al. 2005) in 300 men and women performing graded
treadmill exercise to exhaustion showing that maxi-
mal fat oxidation (MFO) occurs at a workload equal to
48%VO2max, although there was widespread variability
in this measure across individuals. Identifying the ratio
of fat and CHO use in response to exercise training seems
paramount considering the altered metabolism of these
substrates in diabetes (Kelley and Simoneau 1994) and
obesity (Colberg et al. 1995).
Increased capacity for FatOx consistently reported with
endurance training has also been documented in response
to completion of high-intensity interval training (HIIT). In
a seminal study, Burgomaster et  al. (2008) reported simi-
lar increases in whole-body FatOx after 6 weeks of endur-
ance training versus Wingate-based sprint interval train-
ing (SIT). Subsequent studies also identified significant
increases in FatOx in women undergoing submaximal
HIIT (Talanian et al. 2007; Perry et al. 2008). Mechanisms
responsible for these results include increased β-hydroxyl-
CoA-dehydrogenase (β-HAD) activity and fatty acid-bind-
ing protein content shown in response to HIIT (Talanian
et al. 2007; Perry et al. 2008). However, not all data exhibit
improved FatOx in response to SIT (Larsen et  al. 2015).
In addition, recent findings (Astorino and Schubert 2014)
demonstrate that only 65% of participants completing HIIT
or SIT reveal significant increases in FatOx, which suggest
that it may not be improved in all participants completing
chronic interval training.
It is evident that women oxidize more fat at the same
absolute and relative intensity versus men (Cheneviere
et al. 2011) and express MFO at a higher intensity (Vena-
bles et  al. 2005). However, potential differences in FatOx
between men and women in response to HIIT are poorly
understood. No differences in magnitude of reduction in
RER, signifying increased FatOx, were reported in men
and women matched for age and cardiorespiratory fitness
who completed 6 days of Wingate-based SIT (Astorino
et  al. 2011), which supports data from a recent study
(Bagley et al. 2016). Nevertheless, Gillen et al. (2014) sug-
gested that women may respond differently to SIT com-
pared to men. Potential differences in training responses
between genders are due to women’s greater type I muscle
fiber expression (Simoneau et  al. 1985) as well as greater
fat mass and lower fat free mass (Bijlsma et al. 2013) com-
pared to men. Overall, understanding potential gender dif-
ferences in responses to HIIT will better enable clinicians
13
Eur J Appl Physiol
and scientists to tailor exercise programming to various
individuals.
The primary aim of the current study was to compare
changes in MFO and capacity for FatOx amongst different
HIIT regimes, and in addition, to examine potential gender
differences in effects of HIIT in men and women. Unlike
the previous studies in this area (Talanian et al. 2007; Perry
et al. 2008; Bagley et al. 2016), we employed a non-exercis-
ing control group to examine if training-induced differences
in FatOx are truly due to HIIT and not a product of day-to-
day variability in the measure, which is quite large (Croci
et  al. 2014a). It is possible that greater inherent variabil-
ity would make it more difficult to detect statistically sig-
nificant changes owing to an intervention, such as exercise
training. Moreover, participants underwent various regimes
of HIIT, which, to our knowledge, has not been done in the
previous studies investigating changes in FatOx in men and
women. This approach caters to the unique nature of HIIT
whose structure promotes numerous permutations in inten-
sity, duration, or recovery. It was hypothesized that MFO
would be increased in response to HIIT versus non-exercis-
ing controls, with similar increases in MFO evident in all
regimes, and that no differences in the FatOx response to
training would occur between men and women.
Methods
Design
After an overnight fast (12  h) and 48  h abstention from
exercise, participants completed graded cycling until
exhaustion to assess fat and carbohydrate oxidation. At
least 48–72  h later at the same time of day, participants
initiated ten sessions of HIIT during which overload was
instituted after the 3rd and 6th sessions. Forty eight to
96  h after the 10th and final session of HIIT, the graded
cycling test was repeated to measure changes in FatOx
and CHOOx. Participants recorded their habitual physical
activity and dietary intake during the study in a written log.
All sessions were performed in a temperature-controlled
laboratory (temperature and relative humidity = 20–23  °C
and 30–50%, respectively).
Participants
In response to word-of-mouth and electronic bulletins
posted between January 2014 and February 2016, 192 indi-
viduals contacted the Primary Investigator regarding the
study. Of these prospective participants, 77 enrolled in the
study, 43 who initiated HIIT, and 34 non-exercising con-
trols (CON) who met all inclusion criteria but lacked the
time to initiate HIIT. Physical characteristics of participants
Eur J Appl Physiol

who completed all requirements of the study are shown in
Table 1. Participant ethnicity included Caucasian (n = 41),
Hispanic (n = 24), African American (n = 2), Asian/Filipino
(n = 7), and Middle Eastern (n = 3) and reflected the ethnic
composition of the University community. All performed a
minimum of 150  min/week of physical activity, including
resistance training, non-competitive sport, aerobic exer-
cise, CrossFit, surfing, etc., which was confirmed with a
questionnaire (National Cancer Institute 2013). None had
completed regular cycling or interval training in the pre-
ceding 6 months. Inclusion criteria included healthy, body
mass index <30 kg/m2, weight stable in the last 12 months,
non-smoker, as well as lack of knee pain and medication/
supplement use which may modify study outcomes. All
women were eumenorrheic. Written informed consent was
obtained from all participants before initiating the study,
whose procedures were approved by the University Institu-
tional Review Board. During the study, four stopped train-
ing due to lack of time (n = 2), unrelated injury (n = 1), and
failure to adhere to inclusion criteria (n = 1), and two con-
trols did not complete the follow-up trial due to cessation of
habitual exercise outside the study.
Measures
Height and body mass were measured with a scale and sta-
diometer, and body density was measured using skinfolds
recorded at the abdomen, thigh, and chest (men) and tri-
ceps, thigh, and suprailiac (women) following standard-
ized procedures (Jackson and Pollock 1978; Jackson et al.
1980). A blood sample was obtained in duplicate via capil-
lary puncture of a fingertip to measure blood glucose con-
centration (AccuCheck Compact Plus, Roche Diagnostics,
Indianapolis, IN), which was performed to ensure that sub-
jects were fasted. After 5 min of resting gas exchange data
were obtained to yield basal values of ­VO2 (3–4 mL/kg/
min) and respiratory exchange ratio (RER) (<0.85), partici-
pants initiated progressive cycling on an electrically braked
cycle ergometer (Velotron DynaFit Pro, RacerMate, Seat-
tle, WA) consisting of 7 min at 30 (women) or 40 W (men)
followed by 20  W/min increases in power output every
3  min until mean RER was greater than 1.0 for an entire
stage, after which power output was increased by 20 W/min
until voluntary exhaustion and attainment of V ­ O2max and
peak power output (PPO) (Achten et al. 2002). Achten et al.
(2002) demonstrated that in healthy individuals, 3  min
stages are sufficient to attain a steady state. ­VO2max was
determined as the mean of the two highest values attained
during exercise from any 30 s period. A protocol with 3 min
stages was shown to elicit accurate values of ­ VO2peak
(Bishop et al. 1998), and preliminary data in 5 active men
and women showed similar ­VO2max between this protocol
and ramp exercise to exhaustion. During exercise, heart rate
(HR) was assessed via telemetry (Polar Electro, Woodbury,
NY) and gas exchange data were obtained every 15 s using
a metabolic cart (ParvoMedics TrueOne, Sandy, UT) which
was calibrated before exercise according to the manufac-
turer’s recommendation. Gas exchange data were visually
inspected, and any values differing by more than two stand-
ard deviations from the mean value were removed from
subsequent analysis.
During progressive exercise, estimates of oxygen uptake
­(VO2) and carbon dioxide production (­VCO2) were aver-
aged from the last 60 s of each stage to determine RER and
rates of FatOx and CHOOx (Frayn 1983), assuming that
the urinary nitrogen excretion rate was negligible. Data are
derived from five stages of progressive exercise at which
mean RER did not surpass 1.0 culminating at work rates
equal to 110 (women) and 120 W (men). These were com-
pleted by all participants pre- and post-training. This peak
intensity elicited approximately 45%PPO and 57%VO2max,
which would enable accurate calculations of fat and

Table 1  Baseline participant Parameter HIIT + SIT HIIT + HIITHI HIIT + PER CON

characteristics (mean ± SD)
Age (years) 22.5 ± 5.8 21.9 ± 1.9 23.1 ± 5.5 25.8 ± 5.8*
Gender (n = M/F) 7/7 7/6 5/7 15/17
Height (cm) 172.0 ± 7.8 174.4 ± 9.6 170.9 ± 9.9 172.2 ± 11.3
Mass (kg) 68.5 ± 10.3 69.6 ± 11.4 68.2 ± 7.0 72.2 ± 14.3
Body fat (%) 14.9 ± 8.1 14.8 ± 6.3 16.6 ± 6.3 15.9 ± 5.6
FPG (mg/dL) 85.8 ± 8.7 86.5 ± 8.8 86.6 ± 8.3 88.8 ± 9.2
VO2max (mL/kg/min) 39.6 ± 6.8 41.1 ± 4.9 39.5 ± 5.3 40.5 ± 4.9
PPO (W) 226.9 ± 36.6 240.0 ± 34.4 229.2 ± 51.7 243.0 ± 40.9
PA (h/week) 6.7 ± 2.3 5.5 ± 2.1 6.0 ± 2.4 6.2 ± 2.4

HIIT high-intensity interval training, SIT sprint interval training, HIITHI high-volume high-intensity inter-
val training, PER periodized high-intensity interval training, CON control, M male, F female, FBG fasting
blood glucose, PPO peak power output, PA physical activity
*p < 0.05 versus HIIT groups

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 Eur J Appl Physiol

carbohydrate use from gas exchange data. Maximal fat oxi-
dation (MFO) represented the highest rate of fat oxidation
and was reported in g/min, at an absolute power output, and
as a percentage of ­VO2max, HRmax, and PPO. Intraclass
correlation coefficient for repeated determinations of MFO
is equal to 0.82 and the standard error of the mean is equal
to 0.045 g/min. Fatzone (g/min) (Astorino et al. 2013) was
the mean FatOx determined from the MFO and two work
rates closest to MFO, and minimum fat oxidation (Fat-
min) was identified as the work rate (in W) at which mean
RER > 1.0 for the entire stage.
High‑intensity interval training
At least 48  h after baseline testing, participants initi-
ated ten sessions of progressive low-volume HIIT on
the same electrically braked cycle ergometer consisting
of 8–10  min of training per day (Table  2). During the
remaining ten sessions, participants were randomized
using a Latin Squares design (Devillers and Hall 2006) to
one of three regimes: sprint interval training (HIIT + SIT)
consisting of 8–12 sprints during which participants
were required to pedal maximally, high-volume interval
training (HIIT + HIITHI) consisting of repeated 2.5  min
bouts of cycling with 60  s recovery, leading to training
duration equal to 12.5–17.5 min per day, or a periodized
regime (HIIT + PER) consisting of three sessions of high-
volume HIIT, three sessions of SIT, and four sessions of
low-volume HIIT during which training duration varied
from 5 to 15 min per session (Table 2).
A 5  min warm-up at 20%PPO was completed before
training, and active recovery was performed at this work
rate between bouts. During the initial ten sessions, over-
load was implemented every three sessions by incorpo-
rating an additional 1–2 sessions and increasing work rate
by 10%PPO. Participants were given additional recovery
when requested. All exercise training was supervised and
held at the same time of day (±1  h) within participants.
During HIIT, heart rate was determined using telemetry
(Polar Electro, Woodbury, NY) to identify peak (mean
score of all values recorded at the end of each bout) and
session HR, and strong verbal encouragement was pro-
vided. Average work rate (in W) was also recorded using
the Velotron software. Training was performed 3 days/
weeks and at least 24  h was provided between sessions,

Table 2  Description of 20 Training regimes Number of Bout duration Intensity (% Recovery (s) Session
sessions of high-intensity bouts (s) PPO) duration
interval training performed in (min)
the study
HIIT + SIT
 1–3a 8 60 90 75 23.0
 4–6 9 60 100 75 25.25
 7–10 10 60 110 75 27.5
 11–13 8 30 130 120 25.0
 14–16 10 30 140 120 30.0
 17–20 12 30 150 120 35.0
HIIT + HIITHI
 1–3 8 60 90 75 23.0
 4–6 9 60 100 75 25.25
 7–10 10 60 110 75 27.5
 11–13 5 150 70 60 22.5
 14–16 6 150 75 60 26.0
 17–20 7 150 80 60 29.5
HIIT + PER
 1–3 8 60 90 75 23.0
 4–6 9 60 100 75 25.25
 7–10 10 60 110 75 27.5
 11–13 6 150 70 60 26.0
 14–16 10 30 140 120 30.0
 17–20 8 60 100 75 23.0

VO2max was determined after session 10 with PPO from this bout used to set intensity of sessions 11–20
s seconds, PPO peak power output, HIIT high-intensity interval training, HIITHI high-volume high-inten-
sity interval training, SIT sprint interval training
a
 Specific days of HIIT sessions

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Eur J Appl Physiol
which were held on Monday/Wednesday/Friday, Monday/
Tuesday/Thursday, and Tuesday/Thursday/Friday.
Habitual physical activity
Participants were advised to maintain their habitual physi-
cal activity during the study. They were given a training
log upon study initiation and were required to denote daily
physical activity in this log, which was submitted at study
termination to describe volume of regular physical activity
(h/week) completed during the study.
Assessment of dietary intake
Prior to the initial trial, participants completed a 24 h die-
tary recall requiring participants to list not only all food/
drink intake during this period, but also specific time of
day of nutrient ingestion, and this pattern was repeated 24 h
before subsequent assessments. In addition, they tracked
their dietary intake for 4 consecutive days (including 2
weekend days) pre- and post-training, which was analyzed
using commercially available software (the US Department
of Agriculture Nutrient Database; http//ndb.nal.usda.gov/
ndb/foods/list). From baseline to post-HIIT, mean energy
(p = 0.79), fat (p = 0.11), CHO (p = 0.54) and protein intake
(p = 0.91) did not differ, and there were no differences
among groups (p > 0.10) (Table 2).
Statistical analyses
Data are presented as mean ± standard deviation (SD) and
were analyzed with SPSS Version 20.0 (Chicago, IL).
The Shapiro–Wilks test was used to assess normality, and
all variables related to substrate oxidation were found to
be normally distributed (p value >0.52). Differences in
MFO, Fatmin, and Fatzone over time (baseline, 10, and
20 sessions of HIIT) were identified using the analysis of
Fig. 1  Change in heart rate
(mean ± SD) in response to
20 sessions of high-intensity
interval training. *p < 0.05
versus sessions High-intensity
interval training is separated
into Phase 1 (sessions 1–3),
Phase 2 (sessions 4–6), Phase 3
(7–10), Phase 4 (11–13), Phase
5 (14–16), and Phase 6 (17–20)
variance (ANOVA) with repeated measures, with regimen
(4 levels = three HIIT regimens and control) and gender
as between subjects factors. Three-way repeated-measures
ANOVA with between (4 levels = three HIIT regimens and
CON; 5 levels of exercise stage represented as %PPO) and
within-subjects factor (baseline, 10, and 20 sessions of
HIIT) was also used to examine differences in FatOx and
CHOOx. One-way ANOVA was used to determine differ-
ences in baseline physical characteristics between groups.
The Greenhouse–Geisser correction was used to account
for the sphericity assumption of unequal variances across
groups. Tukey’s post hoc test was used to locate differences
between means when a significant F ratio was obtained.
Partial eta-squared (η2p) was used as an estimate of effect
size. Sample size in each group originated from the pre-
vious studies (Burgomaster et al. 2008; Lanzi et al. 2015)
comparing changes in FatOx between different exercise
regimes. Statistical significance was set as p < 0.05.
Results
Training fidelity
Training compliance was equal to 778/780 (99.7% of all
sessions). To verify the intensity of HIIT (Taylor et  al.
2015), average peak HR in response to training is shown in
Fig. 1. Heart rate increased during training (p < 0.001, η2p =
0.25) but there was no timeXregimen interaction (p = 0.41).
Heart rate significantly increased (p < 0.05) from ses-
sions 1–3 to 4–6 and 7–10, when it peaked at 91.0 ± 2.6%
of baseline HRmax. Heart rate declined (p < 0.05) in ses-
sions 11–13 and was similar during the last seven ses-
sions of training compared to values from sessions 1–10.
Average work rate during HIIT increased across time
(p < 0.001, η2p = 0.36) and a significant timeXregimen
interaction (p < 0.001, η2p = 0.43) was shown. During the
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 Eur J Appl Physiol

first ten sessions of HIIT, work rate increased every three

HIIT high-intensity interval training, SIT sprint interval training, HIITHI high-volume high-intensity interval training, PER periodized high-intensity interval training, CON controls, MFO maxi-
0.31 ± 0.08

56.5 ± 28.2
57.0 ± 10.0
23.2 ± 10.2

0.27 ± 0.08
5.2 ± 1.2

35.6 ± 8.6
167 ± 37
sessions of HIIT, although it was similar (p > 0.05) across
regimes (115–130  W). Throughout sessions 11–20, par-

Post
ticipants in HIIT + HIITHI were exercising at significantly
higher (p < 0.05) mean power outputs (127–143  W equal

0.32 ± 0.08

52.6 ± 19.5

0.28 ± 0.08
5.4 ± 1.4

57.3 ± 8.2
22.4 ± 8.4
34.8 ± 6.8
165 ± 37
to 52.9–59.6%PPO) versus HIIT + SIT (107–112  W equal
to 47.3–49.6%PPO) and HIIT + PER (109–124 W equal to

CON
Pre
47.6–54.6%PPO).

0.32 ± 0.12

47.4 ± 18.4

0.27 ± 0.10
Changes in maximal fat oxidation

5.8 ± 2.0

55.7 ± 6.4
21.7 ± 6.8
32.0 ± 6.0
167 ± 45
Post
There were no differences in any measure at baseline
between regimes, although absolute MFO was higher

0.32 ± 0.11

65.5 ± 31.7

0.28 ± 0.13
(p < 0.001) yet relative MFO was similar (p = 0.40) in men

6.1 ± 1.6

61.8 ± 7.4
26.9 ± 9.5
38.4 ± 8.2
160 ± 40
versus women. Table 3 demonstrates changes in MFO and

­ atmax in response to 20 sessions of high-intensity interval training (mean ± SD)

mal fat oxidation, FFM fat free mass, Fatmax exercise intensity at which maximal fat oxidation occurs, HR heart rate, PPO peak power output
Mid
related indices of ­ Fatmax, which were combined across
men and women as there was no timeXgender interaction

HIIT + PER
(p = 0.39). There was no change in MFO (p = 0.11) with

0.28 ± 0.08

50.9 ± 16.4

26.6 ± 10.3

0.24 ± 0.09
5.3 ± 1.6

60.3 ± 4.7

32.9 ± 6.6
149 ± 41
time or timeXregimen interaction (p = 0.19). Controls
showed no change in MFO from baseline (0.32 ± 0.08  g/

Pre
min) to post-testing (0.31 ± 0.08  g/min). Similarly, lack
of change (p = 0.28) was shown for relative MFO and no

0.32 ± 0.11

56.7 ± 24.2

0.29 ± 0.10
5.1 ± 1.3

59.3 ± 6.8
21.3 ± 8.4
36.2 ± 7.2
177 ± 41
change was demonstrated in controls (5.38  ± 1.37  mg/
kg FFM/min versus 5.16 ± 1.20  mg/kg FFM/min). ­Fatmax
Post
expressed according to absolute power output (p = 0.19),
%VO2max (p = 0.81), %HRmax (p = 0.15), and %PPO
0.32 ± 0.09

66.7 ± 32.6

25.3 ± 10.2

0.28 ± 0.06
5.3 ± 1.2

60.3 ± 7.2

36.8 ± 7.6
165 ± 30
(p = 0.59) also was unchanged in response to HIIT, and no
changes were evident in controls. There was a main effect
Mid

of time on Fatmin (p < 0.001, η2p = 0.19), yet no timeXreg-

HIIT + HIITHI

imen interaction occurred (p = 0.25). Fatzone increased

0.29 ± 0.13

50.0 ± 17.8

0.25 ± 0.11
4.9 ± 2.0

59.6 ± 4.4
21.3 ± 6.4
34.5 ± 4.9
(p = 0.036, η2p = 0.07) in response to training, but there was

162 ± 35
no significant timeXregimen interaction (p = 0.065, η2p =
Pre

0.11). This variable did not change in controls (0.28 ± 0.08

versus 0.27 ± 0.08 g/min).
0.32 ± 0.10

65.0 ± 27.1

0.28 ± 0.10
Individual responses in MFO are shown in Fig.  2 for
5.6 ± 2.0

59.8 ± 9.2
25.8 ± 9.7
36.2 ± 8.9
169 ± 31

each HIIT regimen. Some participants in HIIT  + SIT

Table 3  Maximal fat oxidation and physiological measures at F

Post

(n = 3), HIIT + HIITHI (n = 5), and HIIT + PER (n = 5)

revealed meaningful increases in MFO from baseline
0.36± 0.08

59.3 ± 20.6

0.32 ± 0.06

to post-training which surpass the minimum difference

6.2 ± 1.3

59.8 ± 8.4
24.7 ± 7.9
36.8 ± 7.7
178 ± 30

(0.12  g/min) calculated from CON who underwent two

Mid

tests separated by 6 weeks. There was a significant inverse

association between baseline MFO and the change in
MFO seen in response to 20 sessions of HIIT, r = −0.46,
0.31 ± 0.09

54.3 ± 21.0

0.27 ± 0.08
HIIT + SIT

5.4 ± 1.3

61.4 ± 7.6
24.0 ± 8.5
38.4 ± 8.0
161 ± 31

 p < 0.05 at baseline between genders

p = 0.004.
Fat oxidation data combined across gender are shown
Pre

in Fig. 3a–d. At baseline during progressive exercise, there

was no difference in FatOx between regimes (p > 0.05).
MFO (mg/kg FFM/min)

There was a significant main effect of training on FatOx

Fatmax (%VO2max)
Fatmax (%HRmax)a

(p = 0.03, η2p = 0.08) as well as training × regimen interac-

Fatzone (g/min)a
Fatmax (%PPO)

Fatmin (Watt)a

tion (p = 0.045, η2p = 0.10) and training × regimen × stage

MFO (g/min)a

Fatmax (Watt)a

interaction (p = 0.03, η2p = 0.09). Although FatOx was con-

sistently higher in response to all HIIT regimes compared to
baseline, post hoc analyses showed no difference between
a

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Eur J Appl Physiol
Fig. 2  Individual changes in MFO (g/min) in response to 20 ses-
sions of a HIIT + SIT, b HIIT + HIITHI, and c HIIT + PER. 0 = pre-
training value, 10 = value recorded after initial ten training sessions,
and 20 = post-training value. Thin lines are individual participants
and thick line is the mean change in MFO across participants within
regime
means. Data showed that CHOOx increased in response
to training (p = 0.025, η2p = 0.08) but no trainingXregi-
men interaction was exhibited (p = 0.31). Data showed that
CHOOx was consistently lowered via HIIT, but this differ-
ence was not significant versus controls (Fig. 4a–d).
Changes in ­VO2max and peak power output
At baseline, these indices did not differ (p > 0.05) between
regimens, although PPO was higher in men versus
women. There was a significant main effect of training on
­VO2max (p < 0.001, η2p = 0.62) as well as a trainingXregi-
men interaction (p < 0.001, η2p = 0.51), as increases in
­VO2max were shown in HIIT + SIT (39.8 ± 7.3–43.6 ± 6.1
mL/kg/min), HIIT + HIITHI (41.1 ± 4.9–44.6 ± 7.0 mL/
kg/min), and HIIT + PER (39.5 ± 5.6–44.1 ± 5.4 mL/
kg/min) but not in CON (40.4  ± 5.1–40.2  ± 5.5 mL/
kg/min). Peak power output did not change in CON
(243 ± 40–244 ± 41  W) but was significantly increased
(p < 0.001) in HIIT + SIT (230 ± 35–252 ± 39  W),
HIIT + HIITHI (240 ± 34–265 ± 47  W), and HIIT + PER
(229 ± 56–249 ± 53 W).
Discussion
Contrary to our hypothesis, data show no significant dif-
ference in MFO in response to 20 sessions of HIIT vary-
ing in intensity and structure compared to non-exercising
controls, although similar responses occurred between gen-
ders. Nevertheless, small increases in fat oxidation during
progressive exercise were revealed with training (4.3  g),
although this adaptation may not be clinically meaningful.
Individual responses in FatOx to HIIT occurred, as 33% of
participants were “responders” to training. Maximal oxy-
gen uptake was significantly increased in response to HIIT,
which suggests that improvements in maximal capacity
for fat oxidation are not always coincident with improve-
ments in cardiorespiratory fitness. Overall, additional stud-
ies examining changes in FatOx in response to HIIT are
merited, especially at higher exercise intensities which have
limitations when interpreting RER as a measure of sub-
strate oxidation.
Average MFO determined in this study (0.30  g/min
and 5.2  mg/kg FFM/min) is comparable to values previ-
ously reported in the literature in healthy adults complet-
ing progressive cycle ergometry (0.28–0.30  g/min, Croci
et al. 2014a). Bagley et al. (2016) reported MFO equal to
0.36  g/min and 5.1  mg/kg FFM/min in adults, which was
obtained from a graded exercise protocol using larger
work rate increments per stage (30–50  W/3  min) versus
those used in this study. In active men of similar fitness
but higher body fat and age, Croci et al. (2014b) reported
higher absolute (0.40  g/min) and relative MFO (6.1  mg/
kg FFM/min). Although our absolute MFO value is lower
than norms provided by Venables et al. (2005) (0.46 g/min
and 7.8 mg/kg FFM/min), it occurred at similar fractions of
HRmax (60%HRmax versus 62%HRmax). However, Ven-
ables et  al. (2005) required progressive treadmill exercise
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 Eur J Appl Physiol

Fig. 3  Change in fat oxidation (mean ± SD) in response to 20 sessions of a HIIT + SIT, b HIIT + HIITHI, c HIIT + PER, and d in non-exercising
controls. 0 = pre-training value, 10 = value recorded after initial ten training sessions, and 20 = post-training value

Fig. 4  Change in carbohydrate oxidation (mean ± SD) in response to 20 sessions of a HIIT + SIT, b HIIT + HIITHI, c HIIT + PER, and d in non-
exercising controls. 0 = pre-training value, 10 = value recorded after initial ten training sessions, and 20 = post-training value

which has been shown to elicit higher FatOx compared to by as much as 20%. In addition, the majority of studies
cycle ergometry in trained individuals (Capostagno and showing higher MFO were conducted in populations with
Bosch 2010) as well as untrained women (King et al. 2016) ­VO2max between 45 and 50 mL/kg/min (Venables et  al.

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Eur J Appl Physiol
2005), 50–55 mL/kg/min (Croci et  al. 2014b), and >70
mL/kg/min (Van Loon et  al. 2001), whose participants
likely have enhanced oxidative capacity. Similar low rates
of FatOx were revealed in untrained men (­VO2peak = 38.6
mL/kg/min) whose fat oxidation peaked at 22%VO2peak,
after which it did not change when intensity was increased
to 40%VO2peak (Bergman and Brooks 1999). This non-
bell-shaped pattern of FatOx was also shown in our partici-
pants during the study, as FatOx did not differ from stage
1 (30–40  W = 15%PPO) to stage 2 (50–60  W = 20%PPO)
of graded exercise after which it slightly declined with fur-
ther increases in work rate (Fig. 3). Although speculative,
this is likely due to the low V
­ O2max of participants (62%
of participants with V
­ O2max below our mean value = 40.1
mL/kg/min) as well as low amount of body fat (15.4%), as
­VO2max and body fat are positively associated with MFO
(Venables et al. 2005).
Our results refute the previous findings showing
improvements in FatOx after various HIIT regimes.
In active women (Talanian et  al. 2007), seven sessions
of high-volume HIIT consisting of ten 4  min bouts at
90%VO2max reduced RER during 60  min of cycling at
60%VO2max leading to 10–20% increases in whole-body
FatOx. When this protocol was repeated in active men
and women for 6 weeks, similar reductions in RER (−0.02
units) were shown leading to significant increases in FatOx
(Perry et al. 2008). Our data (Fig. 3a) show similar magni-
tude of reductions in RER post-training compared to base-
line, but these were not significant versus CON. In fact,
when CON participants were not included in our analysis,
a significant main effect of time (pre- to post-training) on
MFO and FatOx was revealed, which suggests that capacity
for fat oxidation is improved with HIIT. This result empha-
sizes the need for scientists to compare changes in FatOx
in response to training to a non-exercising control group
rather than to baseline values or to a group of participants
performing continuous exercise training (Lanzi et al. 2015).
Another reason why FatOx was potentially not increased
in this study is the marked variability in this measure.
Croci et al. (2014a) reported coefficient of variation rang-
ing from 16 to 21% for MFO determined from two progres-
sive exercise protocols completed 3–7 days apart. In addi-
tion, the CV for exercise FatOx was considerable (24–49%)
and higher than that reported for CHOOx (8–13%). In this
study, data from CON completing the identical progressive
exercise test 6 weeks apart led to a CV for MFO equal to
25%. However, these values are higher than those reported
in other investigations (10%, Achten et al. 2002; 7%, DeS-
ouza Silveira et  al. 2016). The primary factor responsible
for the variation in FatOx is biological error and likely
not measurement error, which is relatively small for the
metabolic system used in the current study (1.6, 0.04, and
0.04 L min for exercise ­VE, ­VO2, and ­VCO2, respectively).
In fact, Croci et al. (2014a) suggested that dietary intake be
standardized for 48 h before testing to potentially minimize
fluctuations in muscle glycogen content, fat intake, and free
fatty acid concentration, which are determinants of exercise
RER (Goedecke et al. 2000).
An alternative explanation explaining the lack of
changes in FatOx comes from Larsen et  al. (2015), who
postulated that increased capacity for FatOx may primar-
ily occur in response to higher volume HIIT training as
completed in the previous studies (Talanian et  al. 2007;
Perry et  al. 2008) in which there was increased activity
of β–HAD and fatty acid-binding protein content. In con-
trast, participants in the Larsen et  al. (2015) study com-
pleted 5 min/d of SIT at 128%PPO, and results showed no
change in FatOx or these metabolic measures. Training in
this study ranged from as little as 4 min/d of SIT to as high
as 17.5  min of HIIT, which are at minimum 50% lower
than the daily volume of training completed in the stud-
ies by Perry et  al. (2008) and Talanian et  al. (2007). Car-
nitine palmitoyltransferase-1 sensitivity was also enhanced
in response to 8 weeks of endurance training (Bruce et al.
2006) but not after low-volume SIT (Larsen et  al. 2015),
further emphasizing that the overall training volume may
be an important factor to consider when increasing FatOx
is a primary outcome of exercise training.
Figure  2 exhibits that a few individuals showed mean-
ingful increases in MFO in response to training. An inverse
association was shown between baseline MFO and the
magnitude of change with training, suggesting that individ-
uals with low initial FatOx may be more likely to enhance
capacity for fat oxidation in response to low-volume HIIT,
whereas those with relatively high initial rates of fat oxi-
dation during cycling may be relatively unresponsive and
potentially a higher volume or different modality of train-
ing would be necessary to increase fat utilization. Recent
findings (Astorino and Schubert 2014) corroborate this
individual variability by showing that increases in FatOx
occur in approximately 60–65% of men and women per-
forming 6 days of Wingate-based SIT or 12 weeks of aero-
bic interval training. Individual non-response has also been
identified for the change in ­VO2max in response to train-
ing (Bouchard et al. 1999) and highlights the importance of
individualizing exercise programming to each client.
Our data are limited to active young men and women
completing low-volume HIIT on a cycle ergometer, so
data cannot be applied to other populations performing
differences HIIT regimes. In addition, we used a progres-
sive exercise protocol to assess potential changes in FatOx
rather than a constant load exercise bout as used in the
previous studies (Talanian et al. 2007) showing significant
changes in FatOx after HIIT. One advantage of the progres-
sive protocol is that it allows determination of changes in
FatOx across multiple work rates, including MFO, which
13

is related to insulin sensitivity (Robinson et  al. 2015). In
a previous study (Croci et  al. 2014a), graded exercise
consisted of 10  min at 20%PPO followed by 7.5%PPO
increases in power output rather than using absolute work-
loads as used in the current study. This protocol would be
markedly longer than that employed in this study, which
may elicit slightly different estimates of substrate oxida-
tion. We were only able to analyze gas exchange data from
five stages of progressive cycling up to 110–120 W before
RER surpassed 1.0, so we were unable to examine potential
differences in FatOx at higher intensities, which is unfor-
tunate considering that trained women burn significantly
more fat than untrained women at 120–180 W (Stisen et al.
2006). At intensities where RER approaches 1.0, there is
additional production of ­CO2 through bicarbonate buffering
which presents a methodological challenge to determining
substrate oxidation from gas exchange data. This limita-
tion may lead to a 3–12% underestimation of fat oxidation
during exercise (Rowlands 2005). In the current study, men
and women were used to better generalize our results to
the broader adult population, although this may have led
to greater variability in our data and some non-significant
findings. Interval training was not based on ventilatory
threshold, and in addition, blood lactate concentration was
not measured during the study, which may shed light on
different levels of anaerobiosis experienced by participants
during HIIT. In addition, we did not consider the effects of
the menstrual cycle, as Horton et  al. (2002) reported that
it does not influence substrate metabolism. Finally, estima-
tions of fuel use and especially CHOOx using the Frayn
(1983) equations may be slightly imprecise, as they are
based on blood glucose being the primary CHO fuel rather
than muscle glycogen (Jeukendrup and Wallis 2005).
Conclusions
In active men and women, our data exhibit similar magni-
tude of increases in FatOx and MFO in response to 20 ses-
sions of HIIT as previously reported (Bagley et al. 2016),
but when data were compared to a non-exercising control
group, these changes were not statistically significant.
Large variability in FatOx across days likely explains this
lack of training effect as well as the low-volume nature of
training. We recommend that scientists require participants
to abstain from exercise and standardize dietary intake for
48 h before assessment of FatOx to potentially reduce vari-
ability in the measure. In addition, to overcome methodo-
logical limitations of determining substrate oxidation from
RER approaching 1.0, further study is merited using meas-
ures of plasma free fatty acids, glycerol, and glucose com-
bined with muscle glycogen and intramuscular triglyceride
13
Eur J Appl Physiol
content to elucidate effects of various HIIT regimes on sub-
strate partitioning.
Acknowledgements  The authors thank the participants for their
dedication to the study as well as Samantha Namm, Anthony Fischer,
Kimi Wood, Kayla Snow, Matt Montell, Johnnie Durbin, Ramon Con-
treras, Susy Damian, Sarah Offenbecher, and Jordan Ridings for assis-
tance with data collection.
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