The Influence of Pre-exercise Feeding on Acute Bone Turnover Biomarkers in Response to Physical Activity in Healthy Participants
Phase: NA
Conditions: Osteopenia or Osteoporosis
Sponsor: Sheffield Hallam University

1 
1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
RESEARCH PROTOCOL 
 
Sheffield Hallam University 
 
        Local study number:               Ethics Review ID: ER52048815   
 
Protocol Version and 
Date                  
 
Version 5.0 30th June 2024 
 
 
 
Principal Investigator:  
 
 
 
 
 
 
 
 
 
Dr Jasmine Samvelyan 
Senior Lecturer in Biomedical Science  
 
Faculty of Health, Education, Medicine & 
Social Care 
Anglia Ruskin University School of Medicine 
Bishop Hall Lane 
Chelmsford 
CM1 1SQ 
 
Telephone: +44(0) 1245 683738 
 
Email: jasmine.samvelyan@aru.ac.uk 
 
Co-Investigator:  
Dr Simon Bowles 
Senior Lecturer in Nutrition 
 
Sheffield Hallam University 
City Campus 
Owen Building 
Sheffield,  
S1 1WB 
 
  Email: S.Bowles@shu.ac.uk 
 
 
 
 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
Contents  
SECTION 1: SUMMARY OF PROTOCOL DETAILS 
4 
SECTION 2: RESEARCH QUESTION(S) 
5 
SECTION 3: ABSTRACT 
5 
SECTION 4: AIMS AND OBJECTIVES OF THE STUDY 
5 
SECTION 5: BACKGROUND AND RATIONALE 
6 
5.1 Scientific background and relevance ................................................................................. 6 
5.2 Study rationale .................................................................................................................. 8 
5.3 Clinical relevance .............................................................................................................. 9 
SECTION 6: PLAN OF INVESTIGATION 
9 
6.1. Study design ..................................................................................................................... 9 
6.2 Power calculation ............................................................................................................ 10 
6.3 Study measurements ...................................................................................................... 11 
6.4 Methods ......................................................................................................................... 11 
6.4.1 VO2Max and determination of running speed 
11 
5.4.2 Familiarisation 
12 
6.4.3 Downhill running protocol 
12 
6.4.4 Bone Mineral Density (BMD) measurement 
12 
6.4.5 Bone turnover markers, cortisol, insulin and glucose measurements 
12 
6.4.6 Sample handling 
12 
6.5 Setting for the project ..................................................................................................... 13 
6.6 Participants ..................................................................................................................... 13 
6.7 Recruitment .................................................................................................................... 13 
6.7.2 Inclusion/Exclusion criteria 
14 
6.8 Outcome measures and participant involvement at each visit ......................................... 15 
6.8.1 Summary of participant involvement by study time point 
15 
6.8.2 Summary of biochemical measurements by study visit time point (for both fasted and fed conditions) 17 
6.9 Statistical analysis ........................................................................................................... 17 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
6.9.1 Data collection 
18 
6.10 Project plan ................................................................................................................... 18 
SECTION 7: PROJECT MANAGEMENT 
18 
7.1 Study team ...................................................................................................................... 18 
7.2 Amendments to the protocol .......................................................................................... 19 
SECTION 8: EXPERTISE OF THE RESEARCHER AND ASSOCIATED TEAM 
19 
SECTION 9: ETHICAL ISSUES 
20 
SECTION 10: METHODS FOR DISSEMINATING RESEARCH RESULTS 
22 
SECTION 11: STRATEGY FOR TAKING THE WORK FORWARD 
22 
SECTION 12: COSTING THE PROJECT 
22 
SECTION 13: FUNDING 
23 
SECTION 14: REFERENCES 
23 
SECTION 15: OTHER INFORMATION 
25 
Appendix A: Project Plan ......................................................................................................... 1 
 
 
 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
Section 1: Summary of Protocol Details 
 
 
Title of project:  
The influence of pre-exercise feeding on acute bone turnover biomarkers in response to physical 
activity in healthy participants 
 
1.2  
Sheffield Hallam project reference number:  ER52048815   
 
 
1.3  
Protocol version number and date: 
5.0, 30th June 2024 
 
 
 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
Section 2: Research question(s) 
 
1. Does timing of feeding around a mechanical loading stimulus to the skeleton alter the 
response of bone turnover to exercise? 
 
Section 3: Abstract 
 
The benefits of increased human lifespan depend upon the duration of healthy, independent living; the 
health span. Bone-wasting disorders contribute significantly to loss of independence, frailty, and 
morbidity in older people. Therefore, there is an unmet need globally for lifestyle interventions to reduce 
the likelihood of bone fractures throughout the life-course and with ageing. Although many mechanisms 
are involved in disorders of bone loss, there is no single regulatory pathway and, therefore, there is no 
single treatment available to prevent their occurrence.  
 
We have previously shown that in young 17-week-old mice, 16-hour fasting followed by reintroduction of 
food for 2 hours increased markedly the potency of mechanical loading, which mimics the effect of 
exercise, to induce new cortical bone formation (Samvelyan et al, 2021). Consistent with this finding, 
fasting, and re-feeding increased the response of bone to a loading stimulus that, alone, does not 
stimulate new bone formation in ad-lib fed mice. The hormones, including insulin, that change in response 
to timing of feeding have osteogenic effects that interact with loading-mediated effects. Our findings 
indicate associations between timing of food ingestion and bone adaptation to loading.  
 
Our aim in these studies is to determine whether fasting/feeding interventions alter the effect of 
mechanical loading on bone anabolic activities and increase bone mass. Such non-pharmacological 
lifestyle interventions may benefit skeletal health of humans throughout life-course and in older age.  
Section 4: Aims and objectives of the study 
 
The overall aim of this study is to determine whether an adequate mechanical stimulus to the skeleton 
and the timing of feeding, could improve the balance of bone formation to bone resorption and alter bone 
turnover in young adults. The primary endpoint of this study is the change of the bone resorption 
biomarker C-terminal telopeptide of type-1 collagen (CTX-1) expression between pre-exercise and 2 hours 
following the exercise timepoints.   

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
 
Objectives: 
 
• 
To compare the effect of exercise on bone turnover markers before and after the meals following 
overnight fast in young adult male participants   
• 
To assess the synergistic effects of feeding in addition to exercise on bone formation and 
resorption biomarkers in young adult male participants  
• 
To determine the concurrent role of osteotropic hormone insulin in bone homeostasis in young 
adult male participants 
 
Section 5: Background and rationale 
 
5.1 Scientific background and relevance 
The dramatic increase in human life expectancy in the last 60 years has been driven by reduced deaths of 
people in their 60s and 70s, leading to demographic shifts so that most populations globally are 
characterised by increasing numbers of elderly adults. However, an increasing number of these extra years 
of life are spent in poor health with more years spent in care facilities due to reduced ability to live 
independently. Osteoporosis is a major contributor to loss of independence due to bone fractures and 
resulting hospital treatments lead to significant morbidity. Even after successful fracture treatment, 
independent living is compromised in many patients. While drug treatments reduce consequences of 
osteoporosis significantly, there is a pressing need for non-pharmacological interventions to improve 
bone health across the life-course and to reduce likelihood of age-related bone disease.  
 
The skeleton has protective, mechanical and metabolic roles, providing structural support and acting as a 
site for mineral storage (Seeman et al., 2006). Bone turnover, the cellular machinery responsible for bone 
integrity and strength, is a finely balanced process that is responsive to hormones and mechanical loading.  
 
Physical activity is beneficial for the skeleton acting through the process of functional adaptation, a 
process that is less effective in older animals and humans than younger ones (Samvelyan et al., 2021). In 
addition, older people and those with bone loss, who are already at increased risk of fractures, may be 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
unable to perform exercise that is sufficiently vigorous to strengthen their bones. Therefore, there is a 
need to identify the most effective ways to exercise with maximal benefits to the musculoskeletal system.  
 
Exercise is a non-pharmacological intervention that can modify bone turnover, improve bone health and 
reduce the risk of osteoporosis in later life (Bonnet and Ferrari, 2010). Mechanical loads that are produced 
by exercise change local microenvironments of the canalicular networks within the bone framework via 
dynamic fluid shifts stimulating local osteocytes and ultimately bone turnover (Schaffler et al., 2012; Klein-
Nulend et al., 2013).  
 
The complex and coordinated relationship between the endocrine regulation of energy metabolism, 
adipose tissue, and bone homeostasis has been studied extensively (De Paula et al., 2013). Concentrations 
of hormones, including insulin and amylin, change profoundly in anticipation of, during or after eating 
(preprandial, prandial, and postprandial responses). These hormones have potential anabolic effects on 
bone homeostasis, therefore, synergies between endocrine and mechanical effects could alter the bone 
turnover response to exercise.  
 
The measurement of bone turnover markers, such as procollagen type 1 N-terminal propeptide (P1NP) 
and CTX, allow to assess the dynamic fluctuations in metabolic activity of bone (Delmas et al., 2006). 
Prolonged endurance exercise increases CTX (Guillemant et al., 2004; Hermann et al., 2007; Kerschan-
Schindl et al., 2009; Maïmoun et al., 2006; Scott e al., 2010) but bone formation markers are largely 
unresponsive (Guillemant et al., 2004; Hermann et al., 2007; Maimoun et al., 2006; Scott e al., 2010). This 
suggests that prolonged exercise might result in a transient negative bone remodelling balance and this 
acute response may be implicated in changes in bone mineral density with repeated exercise over time in 
some sub-groups. However, the response of bone turnover markers to exercise is highly variable and 
appears to be modality-, intensity-, age- and sex-specific.  
 
Feeding modulates the metabolic responses to prolonged exercise. The metabolic adaptations to fuel 
supply when fed and with fasting are well studied, however, there is limited consideration of the effect of 
these practices on other biological responses to exercise, such as bone metabolism. Fasting attenuates 
the circadian variation in CTX, a marker of bone resorption, whilst feeding suppresses resting CTX within 
1-hour and concentrations are decreased by around 50% after 2–3 hours (Bjarnason et al., 2002; 
Henriksen et al., 2003; Holst et al., 2007).  In contrast, and in line with the response to exercise, bone 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
formation is largely unresponsive to nutrient ingestion (Bjarnason et al., 2002; Henriksen et al., 2003; 
Holst et al., 2007). Concentrations of hormones including insulin and parathyroid hormone (PTH) change 
profoundly across pre-prandial, prandial, and postprandial states. These hormones have potential 
anabolic effects on bone homeostasis, therefore, synergies between endocrine and mechanical effects 
could alter the bone turnover response to exercise.  
 
Despite the reported independent effects of feeding and exercise on bone metabolism, only a limited 
number of studies in humans have investigated how feeding and fasting practices mediate the effects of 
endurance exercise on bone metabolism, with mixed findings (Borer et al 2019; Scott et al., 2012). Existing 
studies in this area have largely been carried out on athletes (Scott et al., 2012) or populations suffering 
from different disease states, such as T1 and T2 diabetes (Borer et al., 2019). Previous studies have also 
not always used a comparator/control arms (Scott et al., 2012).  
 
The flat running protocol used in previous studies (Scott et al., 2012) may also not be optimal to induce 
enough strain on bone for bone turnover markers to fully respond and therefore in designing the study, 
mechanical skeletal loading will be enhanced by using a downhill treadmill running protocol. The idea 
regarding loading is based on a study where running at 3 m/sec at a 6° decline increased ground reaction 
force (GRF) by 24.3%, while running on a +6° incline reduced it by 22% relative to exercise on level surface 
(Gottschall and Kram, 2005). This proof-of-concept study will investigate the effect of an overnight fast 
versus feeding of a single mixed meal, on the acute changes in bone turnover markers to a bout of 
treadmill running in healthy young adults. 
5.2 Study rationale 
Since dietary interventions are associated with healthy ageing (Kalache et al, 2019), a combination of 
exercise and nutrition may benefit bone health in ageing by utilising synergies between osteotropic 
influences. There is already support for this idea. The data from Samvelyan et al (2021) latest study on 
model organisms provides evidence that the effects of anabolic hormones in increasing bone formation 
in response to mechanical loading may be manipulated by changing timing of feeding in relation to 
exercise imposition (Samvelyan et al, 2021). There are already widespread fasting and caloric restriction 
interventions in use in humans for weight control and for healthy ageing. For instance, the 5:2 diet in 
which food intake is restricted to 500–600 calories on 2 days per week, which could be exploited to 
provide fasting experienced by animal models in Samvelyan et al (2021) study before eating and then 
exercising. It has also been shown that in diabetic postmenopausal women there is a significant increase 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
in bone osteogenic response (defined as the ratio of osteogenic C-terminal propeptide of type I collagen 
(CICP) over bone-resorptive CTX markers) when exercise followed a meal but not when it preceded a meal 
(Borer et al., 2019). Further, bone turnover markers and clinical risk factors can be used to identify patients 
at risk of osteoporotic fracture as well as those who have secondary osteoporosis (Clowes and Eastell, 
2000).  
 
5.3 Clinical relevance  
Osteoporosis is a major contributor to loss of independence due to bone fractures and resulting hospital 
treatments lead to significant morbidity. While pharmacological treatments can reduce consequences of 
osteoporosis, there is a pressing need for non-pharmacological interventions to improve bone health 
across the life-course and to reduce likelihood of age-related bone disease.    
 
This study will allow us to determine whether synergistic potentiating effects on bone metabolism are 
observed in humans in relation to the timing of food ingestion and what recommendations can be made 
to people with respect to food ingestion and exercise to obtain the maximal bone benefits from aerobic 
exercise. Furthermore, if we can identify interventions that potentiate bone's response to aerobic exercise 
in younger adults, then such interventions may have the ability to maximise bone mass in younger adults 
so that with ageing, the additional bone mineral density and improved bone microarchitecture would 
extend the time before osteoporotic “fracture thresholds” are reached. Therefore, understanding the 
bone metabolic response to exercise following fasting and feeding in younger adults is important. 
Section 6: Plan of investigation 
 
6.1. Study design 
In this proof-of-concept study, sixteen young adult men (age: 28 ± 4) will be recruited in good physical 
condition. This will be a three-arm randomised cross-over study with a 1-week washout period between 
visits (Figure 1). Participants will be exercised following a 12-hour overnight fast (FAST) and exercised 90 
minutes after a meal (FED) to test the temporal sensitivity of the loaded bone to absorbed nutrients. 
Participants will also attend a breakfast-only arm (without the exercise protocol). The meal will consist of 
60% carbohydrate, 25% fat and 15% protein.  The exercise will be a downhill running protocol adapted 
from previous studies in this area (Scott, 2012; Borer, 2019). Counterbalancing measures will be put in 
place to prevent and minimise order effects.  

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
 
Participants will undergo a baseline assessment for health history (PAR-Q). Baseline measurements of 
weight, height, BMI and maximal oxygen uptake as well as baseline bone mineral density and body 
composition scans of participants will be performed with a dual energy X-ray absorptiometry (DXA) to 
characterise the study sample.   
 
Figure 1: Study Design 
 
 
6.2 Power calculation 
The primary outcome of this study is the difference in change in CTX concentration between the pre-
exercise and 2-hour post-exercise timepoints between the fasted-exercised and fed-exercised arms. 
Based on changes in CTX from a previous study (Scott et al., 2012), we designated 30% as a minimally 
significant difference and that 30% would be approximately 0.168 µg/l. We calculated a standard 
deviation of 0.2 µg/l based on results from our previous studies with serial measurements of CTX. To 
achieve 80% power for demonstrating this mean difference as statistically significant at the 2.5% two-
sided level requires a total of 16 participants (including 2 additional participants for withdrawals). This 
sample size is in line with that of other studies in this area.  

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
6.3 Study measurements 
The blood samples will be collected (10ml) by cannuala at baseline, immediately pre-exercise, and after 
15 minutes and 45 minutes of exercise and after 2-hours of recovery. The osteogenic response to exercise 
will be determined by measuring a marker of bone formation; P1NP and a marker of resorption; CTX-1.  
 
To assist in making inferences on the possible hormonal interactions with mechanical, cardiovascular, and 
psychological stress in our study, and thus their contribution to changes in bone markers, we will also 
measure plasma concentrations of cortisol; a marker of physiological and psychological stress and because 
of its promotion of bone resorption. Insulin and glucose measurements will allow us to assess the change 
of concentrations in fasted and fed states. Urine samples will be collected at baseline, immediately before 
and after the exercise. Samples will be frozen at -80oC and analysed for bone turnover biomarkers later 
with the permission of participants. 
 
 
 
6.4 Methods 
6.4.1 VO2Max and determination of running speed 
Participants will perform a two-stage graded exercise test on a treadmill to determine the speed of 
downhill running that elicits 70% VO2max. For the first stage, VO2max will be determined via a graded 
exercise test. The test will begin with participants completing a 4-min warmup at 4.8km/h, followed by 1 
minute of passive rest. The graded exercise test will start between 8.0 and 9.6 km/h, with the velocity 
increasing by 1.6km/h every 2 mins until reaching a speed of 14.4km/h. At this point, incline will be 
increased by 2% every 2 mins until the participant reaches volitional exhaustion (Succi et al. 2022). Heart 
rate and rate of perceived exertion (RPE) will be recorded via heart rate monitor (Garmin HRM-Dual, 
Garmin, KS, USA) and RPE Scale (Borg, 1982), to confirm maximal effort. Expired air will be collected and 
the gas exchange will be analysed using the Metelyzer 3B (Cortex, Leipzig, Germany). The equipment will 
be calibrated prior to each test as per manufacturer's directions using standard gases. 
 
The second stage of the treadmill test will be used to determine the running speed that elicits 70% 
VO2peak at a decline of -6%. Participants will start running at the same speed as the first test. Speed will 
be increased by 1km every 2 mins for five stages. Regression analysis will then be performed to determine 
the speed that elicited 70% VO2max. Participants will then rest for 30 minutes. 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
5.4.2 Familiarisation 
Once running speed has been determined, the participants will then be familiarised with the downhill 
running protocol to be used in the experimental visits. This familiarisation visit will expose the participant 
to the eccentric strain and the subsequent muscle damage associated with downhill running (Southall-
Edwards, et al. 2022). As muscle damage can have a detrimental effect on markers of bone formation 
(Huang, et al. 2022), the prior exposure of the running protocol to the participants should blunt the muscle 
damage response in subsequent bouts of downhill running due to the repeated bout effect (Khassetarash, 
et al. 2022). 
6.4.3 Downhill running protocol  
Participants will perform 45 min of treadmill running at 70% VO2max at a -6% slope, one and half hours 
after a standardised breakfast (FED) or fasted (FAST).  Downhill treadmill slope will increase Ground 
Reaction Forces (GRFs) relative to level walking (Gotschall and Kram, 2005).    
6.4.4 Bone Mineral Density (BMD) measurement  
Bone mineral density and whole body composition scans will be performed at the familiarisation visit with 
a dual-energy X-ray absorptiometry (DXA) scanner (Hologic). The lumbar spine (L1 through L4), femoral 
neck, trochanter, and total hip areas will be scanned for determination of areal BMD (g/cm2).  
6.4.5 Bone turnover markers, cortisol, insulin and glucose measurements  
Blood samples (around 10ml) will be collected into serum-separation tubes containing spray-coated silica 
and polymer gel for serum separation (BD Vacutainer venous serum separation tubes: Hemogard, Fisher 
Scientific, UK) for determination of bone turnover markers PINP and CTX-1. Measurements of bone 
turnover markers will be done at Bone Biochemistry Laboratory (The University of Sheffield, UK). 
Measurement of glucose, insulin, and cortisol will be done at the Chemical Chemistry laboratory (Sheffield 
Teaching Hospitals, UK).  
6.4.6 Sample handling 
Samples will be collected into Serum Separating Tubes (SST). Blood in SST tubes will be left to clot for 15-
30 minutes at room temperature and centrifuged at 2000 g for 10 minutes. The serum will be aliquoted 
and stored at -800C until analysis. 
 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
6.5 Setting for the project 
  Oversight of the study will be at Sheffield Hallam University. The study will be conducted at the Advanced 
Wellbeing Research Centre (Sheffield Hallam University, UK). The study will conform to all standards of 
Good Clinical Practice and will be overseen by an experienced academic.  
 
The Principal Investigator is Dr Jasmine Samvelyan (Senior Lecturer in Biomedical Science, Anglia Ruskin 
University School of Medicine) and co-investigators for the study are Dr Simon Bowles (Senior Lecturer in 
Nutrition, Sheffield Hallam University), Professor Eugene McCloskey (The University of Sheffield) and 
Professor Richard Eastell (The University of Sheffield). Professor Craig Sale (Manchester Metropolitan 
University) will provide academic support for study concept and design. Data collection (after appropriate 
training) will be the responsibility of Dr Jasmine Samvelyan registered at Anglia Ruskin University.  
 
6.6 Participants 
Participants will be healthy male adults aged 28 + 4 years.  
6.7 Recruitment 
Participants will be invited to take part in the study by invitation email and by poster. Posters will be 
distributed around Sheffield Hallam City Campus Buildings. Invitation emails will be sent out to Food and 
Nutrition students studying on undergraduate and postgraduate Food and Nutrition courses within the 
Department of Service Sector Management. 
 
Prospective participants will be followed up by telephone or email for screening to make sure that they 
meet the eligibility criteria for the study. Eligible participants will be sent an appointment letter to confirm 
their study visit. 
 
6.7.1 Informed consent 
All participants will give informed consent before enrolment on to the study. Each prospective participant 
will receive a participant information sheet giving details of the study and will have at least 24 hours to 
consider their participation in the study before attending their first study visit to give consent to 
participation. All participants will be given the opportunity to ask questions about the study before giving 
informed consent. Informed consent will be collected of each prospective participant by the principal 
investigator/co-investigator or another appropriately trained member of staff after training and 

14 
1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
observation as assigned by the delegation log. Participants will also have the opportunity to ask any 
questions that they have during any of their study visits.  
 
6.7.2 Inclusion/Exclusion criteria 
 
Inclusion criteria will be:  
● Male (females excluded to remove the interplay between menstrual cycle and bone turnover) 
● Caucasian ethnicity 
● Aged 28 + 4 years 
● Are physically active (meet the UK guidelines for physical activity of at least 150 minutes 
moderate intensity activity and/or at least 75 minutes of vigorous intensity activity per week) 
● Otherwise, healthy, able and willing to participate and provide written informed consent 
 
Exclusion criteria will include: 
● Current smokers 
● Excessive alcohol consumption (max 15 alcohol units/week) 
● Any musculoskeletal injury/disabilities  
● Any conditions known to affect bone metabolism (e.g. uncontrolled hyper-/hypothyroidism, 
hyperparathyroidism, hypo-/hypercalcaemia) or malabsorption syndromes (e.g. Crohn’s disease, 
coeliac disease or inflammatory bowel disease).  
● Taking any medication known to affect bone metabolism (such as glucocorticoids or 
bisphosphonates)  
● Positive Covid-19 test within the last 8 weeks 
● Suffered a fracture in the previous 12 months  
● Sedentary status (see physical activity inclusion criteria above)  
● Have been told by medical professionals that they should not take part in moderate to high 
intensity exercise  
● Should not be a professional athlete or take part in significant competitive recreational activity 
(takes no more than 4 structured exercise sessions per week on average) 
● History of diagnosed eating disorders 
 
 
 
 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
6.8 Outcome measures and participant involvement at each visit 
All participants will be required to attend the Advanced Wellbeing Research Centre (AWRC) on four 
separate occasions.  
 
At the familiarisation visit each participant will give informed consent. Consenting participants will 
complete a PAR-Q, undergo a VO2max test, and DXA scan to measure baseline bone mineral density and 
body composition. Baseline anthropometric measures, including height, weight, and BMI will be taken. 
Participants will be asked to follow a standardised diet three days prior to their second study visit. 
Participants will also be asked to refrain from strenuous physical activity during this period and will be 
asked to record their food and drink consumption in a diet diary.  
 
At the second visit, each participant will arrive after a 12-hour overnight fast.  
Counterbalancing measures will be used to assign participants to either the FASTED-exercised, FED-
exercised or breakfast only conditions. During the FED-exercised or breakfast only arms, a standardised 
breakfast will be given at 8.30am. Exercise in the FED condition will take place 1.5 hours after breakfast 
has been administered (10.00am). When in the fasted condition, participants will also begin exercise at 
10.00am.  
 
Blood samples will be taken by cannulation for bone turnover marker measurement and hormone 
measurement at baseline (8.30am), pre-exercise (immediately before exercise), 15 minutes during 
exercise, 45 minutes (immediately post-exercise) and 2 hours post-exercise. Timings will also be 
consistent in the breakfast-only arm.  
 
At the third and fourth visits, following crossover periods, participants will be assigned to the alternative 
conditions and study measurements repeated as per second visit.  
 
 
6.8.1 Summary of participant involvement by study time point 
 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
Study Time Point 
Familiarisation 
Visit  
First Pre-trial 
stage  
(3 days before 
visit 2) 
Visit 2 
 
Second Pre-
trial stage 
(3 days before 
visit 3) 
Visit 3 
 
Third Pre-trial 
stage 
(3 days before 
visit 3) 
Visit 4 
 
Activity 
 
 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
 
Demographic data  
X 
 
 
 
 
 
 
Anthropometry (Height, 
weight & BMI) 
X 
 
 
 
 
 
 
PAR-Q  
X 
 
 
 
 
 
 
DXA Scan (Hip, Spine 
and Body composition) 
X 
 
 
 
 
 
 
VO2Max Measurement 
X 
 
 
 
 
 
 
Downhill Running 
Familiarisation 
X 
 
 
 
 
 
 
Diet Diary 
 
X 
 
X 
 
X 
 
Refrain from vigorous 
physical activity 
 
X 
 
X 
 
X 
 
Follow standardised 
dietary advice 
 
X 
 
X 
 
X 
 
Breakfast only (no 
exercise) or FED-
exercised or FASTED-
exercised condition 
 
 
X 
 
X 
 
X 
Blood samples for  
biochemical 
measurements 
 
 
X 
 
X 
 
X 
Urine Samples 
 
 
x 
 
x 
 
x 
Participant expenses 
given (£100 gift voucher) 
 
 
 
 
 
 
X 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
6.8.2 Summary of biochemical measurements by study visit time point (for both fasted and 
fed conditions) 
 
Study Visit Time 
Point 
Baseline  
(8.30am) 
Immediately 
Pre-exercise 
(10.30am) 
During 
Exercise 
(15-minutes) 
Immediately 
Post-Exercise 
Completion 
(45-minutes) 
2-hours  
Post-exercise 
 
Biochemical 
measurement 
 
 
 
 
 
CTX 
X 
X 
X 
X 
X 
PINP 
X 
X 
X 
X 
X 
Glucose 
X 
X 
X 
X 
X 
Insulin 
X 
X 
X 
X 
X 
Cortisol 
X 
X 
X 
X 
X 
 
6.9 Statistical analysis 
 
 
  
  
  
 
 
Normality will be assumed if Shapiro-Wilk test is not statistically significant (P> 0.05). Where data is 
normally distributed, the data will be presented as mean and standard deviation (SD). Where data were 
not normally distributed, data will be presented as median and IQR.  
 
Biochemical data will be analysed using a linear, mixed model (LMM), with factors 'condition' and 'time 
point' as fixed effects and 'participants' as a random, within-group effect. The assumptions of the LMM 
(homogeneity of variance and normality) will be investigated by examining the distribution of residuals 
and the pattern of residuals versus fitted values. Where non-normality or non- constant variance are 
observed, a log transformation will be applied to the data so that the assumptions are satisfied.  
 
 
 
 
 
Where there is a significant main effect of Time but no significant Condition x Time interaction, each 
subsequent time point will be compared against ‘Baseline’ using a pooled mean from the three groups 
using Dunnett's test. If the Condition x Time interaction is significant, within each group, each time point 
will be compared against baseline using a Dunnett's test and groups will be compared to each other at all 
time points using the Student Newman-Keuls (SNK) test.  
 
 
 
 
 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
6.9.1 Data collection                           
Electronic source data forms will be used to collate all the collected data for each subject. Members of 
the research team will complete data entry into an electronic case report form from participant 
questioning and measurements. All data from the electronic case report forms and the measurement 
reports will be entered onto the study database (created in Microsoft Access) and the data will be stored 
according to the regulations of the Data Protection Act 2018. Data entry will be checked by a second 
operator at a different time point. Participant consent forms will be signed as a paper copy, scanned, and 
stored electronically. The paper copy will be destroyed. The electronic case report forms, other study 
documents (e.g. study log) and study database and will be protected by the standard University windows 
and network login passwords as well as a database login unique to the study. A full data-management 
plan will be submitted for the Converis ethics review.  An electronic participant enrolment log also will be 
maintained to cross-reference subjects against study code. All samples and data will be linked 
anonymously before analysis. No publications will contain information that will be able to identify 
individual participants. 
6.10 Project plan 
We aim to carry out data collection between October 2024 and March 2025. Biochemical analysis on the 
samples will be performed by the end of 2024.  
 
We will consider the study complete when all participants have completed both study data collection time 
points, all laboratory analyses have been performed, the data entered into a database and checked (i.e. 
database lock), statistical analysis is done and the final report is written.  
Section 7: Project management 
7.1 Study team 
Principal Investigator: Dr Jasmine Samvelyan  
Co-Investigators: Dr Simon Bowles, Professor Eugene McCloskey, Professor Richard Eastell, and Professor 
Craig Sale  
Nutritional supervision: Dr Simon Bowles 
Statistical support: All investigators 
Local laboratory supervision: Brent Robbins (AWRC) 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
All investigators will be responsible for study design and study oversight. Dr Jasmine Samvelyan and Dr 
Simon Bowles will be responsible for the day-to-day running of the study including consent and overseeing 
study visits. Dr Jasmine Samvelyan will be responsible for the running of study visits and data collection. 
The electronic Investigator Site File (ISF) will contain a study delegation log which will list all staff involved 
in the study.  
The PI and co-investigators will meet on a regular basis to review the progress of the study and to ensure 
that the project delivers its intended outcomes on time.  
7.2 Amendments to the protocol 
If it is necessary for the protocol to be amended, the amendment and/or a new version of the study 
protocol will be notified through the Converis system for local approval. If necessary, revised Participant 
Information Sheets will be prepared and approved through Converis before participants are provided with 
this new information and asked to re-consent. 
Section 8: Expertise of the researcher and associated team 
Jasmine Samvelyan, The School of Medicine, Faculty of Health, Education, Medicine and Science (FHEMS), 
Anglia Ruskin University is a Senior Lecturer in Biomedical Science, and Lead of the Musculoskeletal and 
Developmental Biology research group at Medical Technology Research Centre (MTRC) at ARU. She has 
10-year extensive research experience in utilising cellular, molecular, and in vivo approaches to study 
musculoskeletal ageing. Dr Samvelyan has used mechanical loaded murine models to explore associations 
between bone homeostasis and energy metabolism and examine microarchitecture of bone in young 
adult and osteoporotic aged mice and has conceived the original idea of this study. This provides 
compelling evidence that she has the necessary skills, accrued knowledge and motivation to successfully 
carry out the proposed project.  
 
Simon Bowles is a Senior Lecturer in Nutrition and a Registered Nutritionist. He has worked within the 
Food Subject Group at Sheffield Hallam University since 2014 as an associate Lecturer and then from 2018 
as a full-time lecturer. He has worked in clinical research for almost 10 years. He has previously worked 
on a Department of Health policy research programme funded project investigating the effect of obesity 
and age on vitamin D metabolism. He was also Chief Investigator for a double-blinded randomised 
controlled trial, administering large bolus doses of vitamin D to vitamin D deficient postmenopausal 
women in order determine the safety of large bolus doses in older people.  

20 
1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
Eugene McCloskey MD, FRCPI is Professor in Adult bone disease, an acknowledged expert in the fields of 
vertebral fracture definition, osteoporosis epidemiology as well as non-invasive assessments of bone 
strength and fracture risk. Professor McCloskey has published over 450 peer-reviewed articles, book 
chapters, guidelines, and reviews. 
 
Richard Eastell MD, FRCP, FRCPath, FMedSci is a medical doctor and Professor of Bone Metabolism at the 
University of Sheffield. He is currently Director of the Mellanby Centre based at the University of Sheffield. 
Some of his recent contributions have been authorship on key papers describing new treatments for 
osteoporosis, such as tibolone, zoledronic acid, denosumab and lasofoxifene as well as addressing issues 
about safety of medications and provide guidelines to diagnose primary hyperparathyroidism, a common 
disorder resulting in high levels of blood calcium. Richard has published over 550 research papers, his 
work as a clinical investigator was recognised in 2014, by the Frederick C Bartter Award from the American 
Society for Bone and Mineral Research. 
 
Craig Sale, Professor at Manchester Metropolitan University has spent over 20 years investigating the 
impact of exercise and nutrition on health and performance in humans, with a particular focus on the 
triggers for adaptations in bone and muscle. He has worked and published in the areas of bone 
metabolism and health, muscle metabolism and performance, genetics, sports nutrition, broader human 
nutrition, biomechanics, cognitive function, and environmental physiology. He is also committed to 
delivering real world impact of significance and reach through research, having experience of conducting 
research on a range of human participants, including elite level and recreational level athletes, military 
personnel, and members of the general population. 
 
Khawla Ekrayem, is a Medical Doctor from Libya Medical School. She has obtained MSc in Public Health 
From The University of Sheffield and currently is Doctoral candidate at Sheffield Hallam University at 
Biomolecular Sciences Research Centre.  
Section 9: Ethical issues 
The protocol will only be implemented after favourable opinion has been received after ethics review via 
the Converis ethics review system at Sheffield Hallam University. The protocol will also be signed off by 
an approved Radiation Protection Expert appointed by the AWRC.  
 

21 
1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
All study participants will receive an information sheet prior to attending the study visit and will give 
written informed consent at the study visit prior to undergoing the study procedures. Informed consent 
will be obtained in accordance with GCP procedures to ensure confidentiality, and privacy.  
 
The security of the research data will be assured by acting in accordance with the 2018 Data Protection 
Act (General Data Protection regulation). A full data management plan will be submitted with the Converis 
review application. The study will be carried out in compliance with the protocol and GCP procedures as 
described in ICH GCP 1996 and the Declaration of Helsinki concerning medical research in humans (2008) 
are in place to ensure appropriate consent, confidentiality, and privacy.  
 
Risk to the participants is minimal. Blood samples will be taken by trained phlebotomists. Consent to use 
and store the samples will be obtained according to the Human Tissue Act 2004. There are small risks from 
having cannula inserted and blood samples taken.  For most people, cannula insertions for blood draws 
do not cause serious problems; however, they may cause a bruise or a small amount of bleeding or pain 
at the puncture site. Some people may feel faint.  In very rare occasions infection may occur. The total 
volume of blood sampling will be small.  
 
This study requires a single DXA scan at the familiarisation visit with a DXA scanner (Hologic). The exposure 
required by the study is additional to routine clinical care. The total protocol dose is 0.02 mSv. This is 
equivalent to 4 days of average natural background radiation in the UK Ionising radiation can cause cancer 
which manifests itself after many years or decades. The risk of developing cancer as a consequence of 
taking part in this study is <0.001 %, which is very low (See letter from Medical Physics Expert). For 
comparison, the natural lifetime cancer incidence in the general population is about 50%. All users of the 
DXA scanner will be fully trained and Ionising Radiation (Medical Exposure) Regulations (IR(ME)R) 
compliant. The protocol has also been approved by the Clinical Radiation Expert at the AWRC (see CRE 
approval letter).  
 
There is also a small risk of injury to participants during the exercise procedure. There is also a small risk 
of participants feeling faint during the procedure. A first aider will be on site at the AWRC if needed in 
such instances. However, participants will be generally healthy and will need to complete a medical 
screening questionnaire before taking part in the study. Only healthy participants who meet the current 
UK physical activity guidelines will be recruited (participants who have been told by medical professionals 

22 
1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
that they should not take part in moderate to high intensity exercise will be excluded).  Appropriate risk 
assessments will be carried out with risk mitigating controls put in place.  
 
Participant samples will be stored at the AWRC before being transferred to the -800C freezer on the 12th 
Floor of the Owen Building (Food and Nutrition Subject Group) at the end of data collection. Samples will 
be stored here until the completion of the study. Other data will be stored securely in accordance with 
the Data Protection Act 2018 and potentially used for future research (with the consent of participants).  
 
Participants will be informed of their right to withdraw from study at any point.  
 
Section 10: Methods for disseminating research results 
It is intended that the results of this study will be published in a peer-reviewed journal as well as presented 
at national and/or international conferences. The data will be used as preliminary data for external grant 
application.  
 
Section 11: Strategy for taking the work forward  
This study is a pilot study and data collected will be used to form part of a wider programme of the study 
of the synergistic effects of feeding interventions on the response of skeleton to physical activity in young 
and elderly humans including females and the concurrent role of candidate osteotropic hormones in bone 
homeostasis. 
 
Section 12: Costing the project 
The project will be funded through Anglia Ruskin University Higher Education Quality-related Research 
(QR) funding, local SHU fieldwork funding and PhD funding, according to the breakdown below:  
 
Expense 
Amount per 
unit (£) 
Total Cost (£) 
Comments 
Office consumables 
 
£350.00 
 QR funding 
Clinical consumables 
 
£2,500.00 
QR funding 
Participant breakfast 
 
£300.00 
QR Funding 
Technical training (e.g. Canulation training) 
 
£700.00 
SHU Fieldwork funding 
Dr Jasmine Samvelyan  
 
£0.00 
Work planned time 

23 
1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
Dr Simon Bowles 
 
£0.00 
Work planned time + 
SRfR time 
Participant Inconvenience Payments 
£100 
 
£1,600.00 
(16 x 
£100.00) 
SHU Fieldwork Funding 
Biomarker analysis  
(80 measurements x 3) 
 
£5,000.00 
Covered by QR funding 
 
Hormone analysis (insulin and cortisol - 
(100 measurements x 2 each cross-over 
study)) 
 
£6,850.00 
Covered by QR funding 
 
Total 
 
£17,300.00 
 
 
Section 13: Funding  
The project is funded through ARU Higher Education Quality-related Research (QR) funding, and by 
internal SHU Fieldwork Funding. 
 
Section 14: References 
 
Bonnet N, Ferrari SL. Exercise and the skeleton: how it works and what it really does. IBMS Bonekey. 
2010;7(7):235-248. https://doi.org/10.1138/20100453.  
 
Borer KT, Zheng Q, Jafari A, Javadi S, Kernozek T. Nutrient Intake Prior to Exercise Is Necessary for Increased 
Osteogenic Marker Response in Diabetic Postmenopausal Women. Nutrients. 2019; 11(7):1494. 
https://doi.org/10.3390/nu11071494. 
 
Borg, GA. Psychophysical bases of perceived exertion, Medicine and Science in Sports and Exercise, 1982; 14(5), pp. 
377-81. 
 
Bjarnason NH, Henriksen EE, Alexandersen P, Christgau S, Henriksen DB, Christiansen C. Mechanism of circadian 
variation in bone resorption. Bone 2002;30:307-13. 
 
Clowes JA, Eastell R. The role of bone turnover markers and risk factors in the assessment of osteoporosis and 
fracture risk. Best Pract Res Clin Endocrinol Metab. 2000 Jun;14(2):213-32. doi: 10.1053/beem.2000.0070. PMID: 
11035903. 
 
Delmas PD, Eastell R, Garnero P, Seibel MJ, & Stepan J. The use of biochemical markers of bone turnover in 
osteoporosis, Osteoporos. Int. 6 2000; 2–17 (supp). 
 
De Paula FJA, Rosen CJ. Bone remodeling and energy metabolism: new perspectives. Bone Res. 2013;1:72-84. 
https://doi.org/10.4248/BR201301005. 
 
Gottschall JS, Kram R. Ground reaction forces during downhill and uphill running. J. Biomech. 2005; 38, 445–452. 
 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
Guillemant J, Accarie C, Peres G, Guillemant S. Acute effects of an oral calcium load on markers of bone 
metabolism during endurance cycling exercise in male athletes. Calcif Tissue Int 2004;74:407-14. 
 
Henriksen DB, Alexandersen P, Bjarnason NH, Vilsbøll T, Hartmann B, Henriksen EE, et al. Role of gastrointestinal 
hormones in postprandial reduction of bone resorption. J Bone Miner Res 2003;18:2180-9. 
 
Herrmann M, Müller M, Scharhag J, Sand-Hill M, Kindermann W, Herrmann W. The effect of endurance exercise-
induced lactacidosis on biochemical markers of bone turnover. Clin Chem Lab Med 2007;45:1381-9. 
 
Holst JJ, Hartmann B, Gottschalck IB, Jeppesen PB, Miholic J, Henriksen DB. Bone resorption is decreased 
postprandially by intestinal factors and glucagon-like peptide-2 is a possible candidate. Scand J Gastroenterol 
2007;42:814-20. 
 
Huang, TH, Nosaka, K, Chen, TC. Changes in blood bone markers after the first and second bouts of whole-body 
eccentric exercises, Scandinavian Journal of Medicine & Science in Sports, 2022; 32(3), pp. 521-532. 
 
Kalache, A., de Hoogh, A. I., Howlett, S. E., Kennedy, B., Eggersdorfer, M., Marsman, D. S., Shao, A., & Griffiths, J. C. 
Nutrition interventions for healthy ageing across the lifespan: a conference report. European Journal of 
Nutrition, 2019; 58(Suppl 1), 1–11. https://doi.org/10.1007/s00394-019-02027-z 
 
Kerschan-Schindl K, Thalmann M, Sodeck GH, Skenderi K, Matalas AL, Grampp S, et al. A 246-km continuous 
running race causes significant changes in bone metabolism. Bone 2009; 45:1079-83. 
 
Khassetarash, A., Baggaley, M, Vernillo, G, Millet, GY, Edwards, WB. The repeated bout effect influences lower-
extremity biomechanics during a 30-min downhill run, European Journal of Sport Science, 2022; pp. 1-10. 
 
Klein-Nulend J, Bakker AD, Bacabac RG, Vatsa A, Weinbaum A. Mechanosensation and transduction in osteocytes, 
Bone. 54 (2) 2013; 182–190. 
 
Maïmoun L, Manetta J, Couret I, Dupuy AM, Mariano-Goulart D, Micallef JP, et al. The intensity level of physical 
exercise and the bone metabolism response. Int J Sports Med 2006;27:105-11. 
 
Samvelyan HJ, Mathers CJ, Skerry MT, Feeding intervention potentiates the effect of mechanical loading to induce 
new bone formation in mice. The FASEB Journal, 2021; 35:e21792. DOI: 10.1096/fj.202100334RR. 
 
Scott JPR, Sale C, Greeves JP, Casey A, Dutton J, Fraser WD. The effects of training status on the metabolic 
response of bone to an acute bout of exhaustive treadmill running. J Clin Endocrinol Metab 2010; 95:3918-25. 
 
Scott JP, Sale C, Greeves JP, Casey A, Dutton J, Fraser WD.  Effect of fasting versus feeding on the bone metabolic 
response to running. Bone 51(6):990-9. doi: 10.1016/j.bone.2012.08.128. Epub 2012 Aug 30. PMID: 22960044. 
 
Schaffler MB, Kennedy OD. Osteocyte signaling in bone, Curr. Osteoporos. Rep. 10 (2) 2012; 118–125. 
 
Seeman E, Delmas PD. Bone quality—the material and structural basis of bone strength and fragility, N. Engl. J. 
Med. 354 (21) 2006; 2250–2261. 
 
Southall-Edwards, R, Innes, S., Ali, A, Jones, B. The effect of downhill running conditions on muscle damage in 
recreationally active adults, Journal of Human Sport and Exercise, 2022; 17(2), pp. 400-408. 
 
Succi, PJ, Benitez, B, Kwak, M, Bergstrom, HC. Methodological considerations for the determination of VO2max in 
healthy men, European Journal of Applied Physiology, 2022; pp100 

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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
Section 15: Other Information 
 
Location of data collection:  
 
Advanced Wellbeing Research Centre 
Sheffield Hallam University 
Olympic Legacy Park  
2 Old Hall Rd 
S9 3TU 
 
 
Laboratory Details: 
 
Sheffield Laboratory Medicine 
Sheffield Teaching Hospitals NHS Foundation Trust  
Northern General Hospital  
Herries Drive 
Sheffield 
S5 7AU 
 
 
The University of Sheffield  
School of Medicine, Dentistry and Health  
Department of Oncology and Metabolism 
Firth Court  
Western Bank  
Sheffield 
S10 2TN

 
 
 
 
 
 
 
 
 
 
 
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1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024 
 
Appendix A: Project Plan
 
 
 
 
Year 1 
Year 2 
Project Month 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
Date 
Apr 
24 
May 
24 
Jun 
 24 
Jul 
 24 
Aug
24 
Sep 
24 
Oct 
24 
Nov 
24 
Dec 
24 
Jan 
 25 
Feb 
25 
Mar 
25 
Apr 
25 
May 
25 
Jun 
25 
Month relative to recruitment 
start 
-4 
-3 
-2 
-1 
0 
+1 
+2 
+3 
+4 
+5 
+6 
+7 
+8 
+9 
+10 
Activity 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study methodology 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Recruitment strategy 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study Specific SOPs 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study Database 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Project management meetings  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Documentation for Research Ethics 
application 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Submission to REC 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Staff Initiation 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Ethical approval expected 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Expected recruitment start date 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Recruitment 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study visits 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Biochemical measurements 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Data entry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Data cleaning 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Data lock 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Data analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Reports/Abstracts/ 
Manuscripts 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
 
 
 
 
26 
 
1.1.1 Effect of pre-exercise feeding on BTMs v5.0 30th June 2024