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7.1 Introduction

HIIT is a very effective exercise protocol at reducing adiposity and elevating energy expenditure compared to other exercise models (Tremblay et al., 1994; Trapp et al., 2008; Macpherson et al., 2011; Gremeaux et al., 2012). This exercise type has additional health benefits such as improved insulin sensitivity (Little et al., 2011; Gillen et al., 2012) and improved cardiovascular health (Wisloff et al., 2007; Helgerund et al., 2010; Guirand et al.,

2011; Meyer et al., 2012). From a fat loss perspective, it is essential to determine the best HIIE model that maximises fat utilisation and enhances energy expenditure.

Many HIIT models have been employed by various research groups; comparing CON to HIIE and show significant fat mass loss, particularly abdominal fat loss (Trapp et al., 2008) after HIIT compared to CONT (Tremblay et al., 1994; Trapp et al., 2008), demonstrated in section 2.9.1. It is beneficial to maximise the metabolic benefits of HIIT by employing an optimal HIIE protocol which exacerbates each metabolic consideration of reducing adiposity and shifting energy balance negative. Chapter 4 of this thesis demonstrated that out all maximal effort exercise elevated purine loss, but resulted potential depressed fat utilisation. Whereas chapter 5 aimed at elevating purine loss and elevating fat utilisation by manipulating HIIE protocols and established heightened purine loss and possible reliance on fat and glycogen during exercise. However as exercise intensity increased and HIIE models

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manipulated, these metabolic changes were not improved. Chapter 6 of this thesis employed exercise protocols from chapter 4 and 5 and validated another potentially more influential metabolic avenue for energy deficit that is unaccounted for in energy balance, urinary lactate excretion. Therefore it is favourable to combine “all out” maximal intensity exercise with the work and rest HIIE model to construct an exercise model that encompasses all beneficial aspects of the modalities utilised in chapter 4, 5 and 6 of this thesis will be tested in males and females.

Males and females exhibit diverse metabolic responses during rest and exercise conditions, owing to differences in male and female steroid hormone concentrations and fluctuations (D’eon et al., 2012). Hormones influence substrate metabolism, specifically in females, oestrogen and progesterone exhibit antagonistic effects on lipolysis as stated in section 2.10. Moreover females typically show a greater reliance on fat as an energy source compared to males. Hence using exercise protocols aimed at decreasing adiposity, females may lose greater relative amounts of fat mass compared to males. Therefore this study will investigate substrate metabolism in males and females in maximal effort HIIE.

7.1.1 Aims and hypothesis

This study will employ ‘all out cycling’ maximum effort HIIE protocol, to determine whether an ‘all out’ HIIE model will induce all favourable metabolic changes needed to maximise energy deficit observed in the earlier chapters of this thesis. HIIE protocols from another research group will be employed but altered slightly, increasing the exercise intensity to ‘all out’ maximum effort and decreasing exercise duration (Trapp et al., 2007). Commissioning females, Trapp et al. (2007) employed two 20 mins of HIIE, the two

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protocols; 8 s cycling, separated by 12 s rest (8:12) and 24 s cycling followed by 36 s rest

(24:36). Exercise intensity was set at the power output achieved at 70% VO2peak. The current

study will also use the 8:12 and 24:36 model, but increase intensity to wingate levels (resistance set against body weight) and decrease the total HIIE duration to 10mins.

The aim was to investigate the metabolic response and changes in substrate utilisation during these two different all out HIIE models in order to see if either protocol produces favourable shifts in metabolism aimed at producing metabolic markers reflective of decreasing adiposity and enhancing energy expenditure.

It is hypothesised that purine nucleotide loss and lactate excretion will be significantly greater in the 24:36 model as opposed to the 8:12 bout of the male group, reflective of greater ATP degradation and glycolysis thus opportunity for energy loss. In addition to this, as the rest period is 36 s compared to 12 s, possible greater fat utilisation will be observed in the 24:36 HIIE trial. In the female cohort, it ishypothesised that the metabolic patterns will be similar to the males, with the 24:36 HIIE trial inducing significantly greater changes in plasma and urinary markers of metabolism compared to the 8:12 HIIE trial. Although similar patterns in plasma and urine may be found, it is hypothesised that there will be a greater change in plasma glycerol in females compared to males, an indirect reflection of greater possibility of fat utilisation. In addition, plasma and urinary metabolites indicative of purine metabolism will be in lower in relative concentrations in females compared to males as females are better at reamination of Hx.

166 | P a g e 7.2 Methods

7.2.1 Participant characteristics

Eight healthy, untrained males aged between 18-35 years (29 ± 3.8yrs, 177 ± 7.7cm, 77 ± 10kg; 48 ± 5.8 ml.kg.min-1) and sevenhealthy, untrained females aged between 18-35 years volunteered to take part in this study (27 ± 5 yrs.; 165 ± 4.5 cm, 65 ± 8.4 kg; 37±5.5 ml.kg.min-1_{) volunteered to take part in this study The experimental design and all}

procedures undertaken by participants were approved by the Victoria University Human Research Ethics Committee performed in accordance with the ethical standards set out in the 1964 Declaration of Helsinki. Participants provided written and verbal informed consent and completed medical history forms prior to the commencement.

7.2.2 Preliminary Testing

At least one week prior to trial days, all participants underwent a VO2 peak test

comprehensive detailed located in section 3.2 regarding preliminary testing. The W was set different for males and females, the female protocol consisting of 3 min x 3 sub-maximal workloads of 25, 50 and 75 watts (W), each subsequent workload increased every minute by 25 W until volitional exhaustion as detailed in section 3.2.1.

7.2.2.1 Respiratory gas exchange

Respiratory gas measurements are described in section 3.5.1.

7.2.3 Familiarisation session

Participants underwent a familiarisation session prior to the testing days as set out in section 3.3.

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Female participants underwent a familiarisation session prior to the testing days. As trial days were separated by approximately one month (see section 3.4 experimental design), two familiarisation sessions were given to each participant to ensure complete replication of the trial day.

7.2.4 Trial procedure

For 24 hrs prior to the trial testing day, participants were instructed to abstain from alcohol, physical activity and caffeine as well as maintaining a 24 hrs food diary in order to replicate meals for the following trial days to prevent substrate variation.

7.2.4.1 Experimental design

In a randomised order and separated by at least one week for males and one for female participants, completed the two exercise protocols; 8 s all out cycling followed by 12 s rest (8:12) and 24 s all out cycling followed by 36 s rest (24:36).

Exercise bouts were 10 mins duration preceded by a rest period and followed by 90mins of recovery.

Participants arrived at the laboratory in the morning in a post-absorptive state after an overnight fast of approximately 10-12 hrs and were instructed to consume water ad libitum. The start time for each trial for a given participant remained consistent to avoid influence of circadian variance. While resting in a supine position, participants gave rest respiratory gas measures and were prepared for blood collection.

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Respiratory gases were collected at rest and for the first and last 10 mins of the 90 min recovery period. Collection measures are detailed in section 3.5.

7.2.4.3 Blood Collection and Analysis

Blood was collected at rest, at 5 mins of exercise (E5), at the end of exercise (E10) and recurrently during the recovery period at 5 (R5), 10 (R10), 15 (R15), 30 (R30), 45 (R45), 60 (R60), 75 (R75), 90 mins (R90). Blood sampling methods are described in section 3.5.4 with analytical methods of plasma FFA, glycerol, glucose, lactate, insulin, inosine, xanthine, Hx and uric acid found in section 3.5.4.2.

7.2.4.4 Urine collection and analysis

Urine was collected as per details in section 3.5.5. Urine samples were measured for inosine, Hx, xanthine, uric acid and lactate as per plasma analysis section 3.5.5.

7.2.4.5 Rating of perceived exertion

Borg scale rating of perceived exertion was shown at the 5th and 10th mins of exercise as per instruction described in 3.5.3.

7.2.5 Statistical Analysis

Detailed in section 3.6, all results is expressed as means ± SEM except for data pertaining to subject characteristics is expressed as means ± SD. All results was analysed using Graph-pad Prism software version 6.02 and statistical significance was accepted at p <0.05. Two-way repeated measures ANOVA with time as the within group factor and

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exercise protocol as the between group factor was completed to detect significance in all plasma, heart rate power, and respiratory gas results. Where an interaction between trials was detected, post hoc tests were completed as mentioned previously. A matched, paired t- test was performed on urinary results comparing post exercise to basal values and determining if the two recovery values were significantly different.

170 | P a g e 7.3 Results

7.3.1 Peak power

In both male and female groups, peak power decreased over the 10min HIIE bout with peak powers in minute 6 significantly lower than minute 1 (p<0.05) in the male group and lower than minute 1 at minute 3 for both HIIE trials in the female group. No differences were detected between 8:12 and 24:36 HIIE trials.

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Figure 7.0: Peak power (W) of maximal all HIIE, 8:12 compared to 24:36 trials in the male group.

Data presented as mean ± SEM; N = 8 * p<0.05 significantly different from rest

The power outputs from the 3 sprints from the 8:12 protocol were averaged in order for comparisons to the 24:36 protocol.

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Figure 7.1: Peak power (W) of maximal all HIIE, 8:12 compared to 24:36 trials in the female group.

Data presented as mean ± SEM; N = 7 * p<0.05 significantly different from rest

The power outputs from the 3 sprints from the 8:12 protocol were averaged in order for comparisons to the 24:36 protocol.