Implications for methodological design

Protocol design - As discussed previously, it is likely that the participants in Studies

1 and 2 (Chapter 4 and 5) did not exceed the potential ‘muscle glycogen threshold’ that is required to fully activate the regulatory cell signalling pathways during exercise. However, since no muscle biopsy was obtained prior to the afternoon exercise bout in either study it is difficult to determine what level of muscle glycogen concentration was available prior to commencing afternoon HIT. Additionally, the time-course of mRNA production following exercise is not well characterised and it is possible that time-course of biopsies adopted here (i.e. post, 30h and 15-h post PM HIT) was not optimal to capture peak responses. Additionally, while measuring mRNA content is often used to demonstrate the presence of signalling pathways, this does not

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necessarily translate into increased protein abundance (Hornberger et al., 2016). Similarly, while the use of p70S6K1 is a useful marker of signalling within the mTOR pathway, this does not necessarily indicate the presence of MPS despite previous investigations highlighting the requirement of mTOR signalling in MPS responses (Dickinson et al., 2011). Future studies should now assess post-translational modifications and the biological/functional significance of these changes chronically when performed over a longer-term training period to gain better understanding of the precise mechanisms underpinning the muscle adaptive responses which occur in response to training and nutritional stimuli. Indeed, this would give a better indication of how to potentially periodise some of these strategies within an athletes training programme across a cycle for maximum benefit. In study 2b, no significant changes were displayed with leptin in response to exercise, something which is unusual in comparison to previous research. It is likely that the blood sampling frequency here wasn’t enough to pick up these changes in circulating levels, and as such further samples taken during exercise and in the hours following each bout would have been useful. More subjective measures of appetite could also have been included as part of a questionnaire given to participants to gain better understanding of the psychological implications of each of the diets.

Participants – The participants used for the studies in this thesis were recreationally

active male subjects who were free from injury and currently participating in running based activity twice per week. Because of this factor, the training status of individual participants may undoubtedly differ due to the specific nature of their training history (i.e. running, cycling, triathlon etc) and that provides altered adaptive stimuli (i.e. frequency and intensity). As such, it is unclear whether the findings from this thesis

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would be translatable to elite endurance populations given the physiological and metabolic differences that would exist. As this thesis contained large amount of cell- signalling analysis, with training history potentially impacting upon this (Coffey et al., 2006), future studies should pay particular attention to the inclusion criteria for subject recruitment so as to minimise the effect of training status upon cell signalling responses. In addition, only male participants were recruited for the studies in this thesis. It is possible that the responses observed would differ if female participants were included, thus future research should also look to include females for studies of this type.

Dietary interventions – Although all food was provided to participants in both studies

in order to try to maximise compliance, there were still some issues with consumption of the foods chosen in places. For example, the high CHO diets contained large volumes of both food and fluid in order to meet the CHO amounts required, and as such there were some GI issues during the afternoon HIT session. Similarly, some of the meals in the high fat diet contained foods such as oily fish and coconut oil which some participants found difficult to consume immediately following high intensity exercise, so in places these meals were consumed more slowly. Future studies in real world endurance populations should look to make any dietary interventions more specific to each participant based on individual preference and foods they are used to consuming on a day to day basis. Due to the nature of the studies, it was difficult to blind which experimental trial was which, and as such participants were aware of which was the low CHO and low energy trial. Although RPE did not significantly differ between trials, participants in some cases did arrive to the laboratory for the low CHO trials with the perception they would fatigue more. In Study 2b (Chapter 6), energy availability was estimated by calculating the difference between the prescribed

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energy intake, and the energy expenditure during AM HIT and PM HIT exercise. Although all food was administered to participants across each trial, limitations also exist when using indirect calorimetry to assess energy expenditure during high intensity exercise (Jeukendrup and Wallis, 2005). Additionally, the use of indirect calorimetry does not permit the assessment of lipid oxidation from peripheral versus intramuscular stores, as has been examined previously (Hulston et al., 2010). Furthermore, energy availability was estimated from fat free mass (FFM) with the assumption that all participants were ~15% body fat. Since participants were not assessed for body composition using dual energy X-ray absorptiometry (DEXA) prior to participating in the study, estimations of energy availability have obvious limitations.