Trial Review

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Trial registered on ANZCTR


Registration number
ACTRN12618000594224
Ethics application status
Approved
Date submitted
5/04/2018
Date registered
17/04/2018
Date last updated
26/04/2019
Date data sharing statement initially provided
26/04/2019
Type of registration
Prospectively registered

Titles & IDs
Public title
The effect of a 4-week ketogenic diet on immune function in endurance athletes.
Scientific title
The effect of a low carbohydrate, high fat ketogenic diet on T-cell cytokine expression and lymphocyte activation in endurance athletes.
Secondary ID [1] 291786 0
Nil
Universal Trial Number (UTN)
U1111-1211-7601
Trial acronym
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Immune function 307265 0
Condition category
Condition code
Inflammatory and Immune System 306383 306383 0 0
Other inflammatory or immune system disorders
Diet and Nutrition 306445 306445 0 0
Other diet and nutrition disorders

Intervention/exposure
Study type
Interventional
Description of intervention(s) / exposure
Study design: Repeated measures, randomised, counterbalanced, crossover design with a 14 day washout period.

Screening and consent: Participants who respond to initial calls of interest will be screened and educated on the requirements of the study. Participants who are deemed eligible and would like to participate, will be asked to complete and sign a consent form and health and eligibility questionnaire. An initial blood draw will be collected by a trained phlebotomist via venepuncture to the antecubital vein to ensure red blood cell, white blood cell and differential cell counts are normal as part of screening. Following this, participants will be asked to complete the Athlete Diet Index (ADI) Eligible participants will be allocated to either dietary condition (i.e. habitual diet (control) or ketogenic low carbohydrate, high fat diet) in a randomised, crossover design (www.randomizer.org), with dietary conditions counterbalanced.

Testing block:

I. Preliminary test: Participants will be required to complete a 5-day image-assisted weighed dietary record on days -5, -4 and -3 to measure nutrient intake prior to the testing block and on days -2 and -1 to measure nutrient intake prior to the time to exhaustion protocol. A registered dietitian (RD) will educate participants on how to record their dietary intake and will code the dietary data for nutrient analysis. If participants are consuming <40% of their energy intake from carbohydrate, they will be deemed ineligible. Participants will arrive at the laboratory between 06:00 and 07:00 h following an overnight fast (i.e. day -2) and having abstained from caffeine, alcohol and strenuous exercise for the previous 24 h. Participants will be asked to void their bladder before their body mass, height and sum of 8 skinfolds measured by an International Society for the Advancement of Kinanthropometry Level 1 Accredited Anthropometrist. Participants will perform an incremental step test to volitional exhaustion on a motorised treadmill. Participants will walk for 3 min at 7.5km/hr, then run for 3 min stages at 9, 10.5, 12, 13.5 and 15km/hr against a 1% gradient to simulate the energetic cost of level-gradient outdoor running. The expired gas will be collected and analysed continuously using a computerised metabolic system with mixing chamber (Parvo Medics TrueOne 2400, Salt Lake City, Utah, USA). The expired gas will be used to estimate substrate oxidation using stoichiometry, see equations below, by averaging the final 30 sec of each stage. Rate of perceived exertion (RPE; using the Borg scale) will be noted during the final 30 sec of each stage and heart rate (HR) will be continuously measured using short-range telemetry. Following the completion of the 15km/hr stage, treadmill velocity will be reduced to 11km/hr and subsequently increased of 0.5km/hr every 30 sec until the attainment of volition exhaustion. Maximal oxygen uptake (VO2max) will be determined by averaging the highest 30 sec period. Simple regression equations will be used to estimate the speed required to elicit 70% VO2max for the use in the subsequent trial.

Substrate oxidation:
CHO oxidation (g/min) = 4.585 VCO2 – 3.226 VO2
Fat oxidation (g/min) = 1.695 VO2 – 1.701 VCO2

Participants will be required to attain a VO2max of >55ml/kg min to be deemed eligible. Eligible participants will be asked to refrain from strenuous exercise until their experimental trial (i.e. day 0), thus providing 48 hrs of recovery. In order to assess shifts in VO2max and substrate oxidation, participants will be required to perform the preliminary test twice for each dietary allocation (i.e. day -2 and day 29).

II. Education session: Participants will be required to undertake a detailed education session (1 hr) immediately following their preliminary test of each dietary condition covering the dietary, training and specimen collection requirements of the study. All participant queries will be answered and possible barriers will be addressed. Participants will be informed of their dietary allocation to offer sufficient time to prepare. Furthermore, participants will be asked to follow their typical dietary intake as they would prior to a main event for the subsequent 2 days (i.e. day -2 and -1) and consume a standardised breakfast (i.e. 2g/kg of carbohydrate at 06:00) (guidance will be provided by a RD) accompanied by a 500ml of water on the morning of their experimental trial. Participants will be asked to replicate this process between days -2 and 0 for their alternate dietary condition and days 29 and 30 the dietary control condition. For the final experimental trial on the ketogenic diet condition, participants will be required to continue their allocated diet (i.e. ketogenic diet) following their follow up preliminary test and will be prescribed an isocaloric low carbohydrate, high fat breakfast accompanied by 500ml of water to be consumed at 06:00. Participants will be required to record their intake via an image-assisted weighed dietary record using WhatsApp.

III. Experimental trial: Two days following the preliminary test (i.e. day 0), participants will arrive at the laboratory at 07:00 h having consumed their prescribed breakfast (i.e. 2g/kg) at 06:00 and refrained from caffeine and alcohol for the previous 24 h. Participants will collect their first morning void in a plastic container and bring to the laboratory to have urine specific gravity measured.

On arrival, participants will have a cannula inserted into their antecubital vein by a trained phlebotomist for serial blood sampling during the trial. Baseline body mass, venous bloods, ketone (D-BHB) and lactate levels will be collected. Participants will commence running at 07:30 at a speed eliciting 70% of their VO2max until exhaustion. Adjustments will be made to the running speed during the first 10 min based on real time measurements of oxygen uptake. This speed will also be used in the follow up experimental trial at the end of the dietary trial. Participants will be fluid restricted to 4ml/kg every 20 min of either a (7%) carbohydrate-electrolyte drink or an electrolyte-only drink for the final run to exhaustion following the ketogenic diet. During the TTE following the ketogenic diet, participants will consume low carbohydrate, high fat snacks calorie-matched to their carbohydrate intake during the high-carbohydrate trials. Participants will have a ventilatory mask fitted for 4 min to collect expired gas samples every 30 min and at exhaustion, with RPE and HR obtained during the last 30 sec. A venous blood sample (serum vacutainer only) and capillary blood ketones and lactate will be collected at 60 and 120 min and the cannula will be flushed with 3-4 ml of saline every 30 min. On attainment of volition exhaustion, treadmill speed will be reduced to 4.4km/hr for two min, then restored to the speed eliciting 70% VO2max and the participant will again run to exhaustion. This process will be repeated so at the third attainment of volitional exhaustion the test will be terminated. This protocol has lower coefficient of variation for exercise capacity compared to traditional single exhaustion protocols. Body mass, venous bloods, ketones and lactate levels will be collected immediately upon termination of the test. The same researcher will monitor each experimental trial. Standardised feedback will be provided and the participants will be encouraged with a financial incentive (i.e. longest accumulated time across all four time to exhaustion tests). Participants will not be notified of their results until the end of all experimental trials. Participants will be provided with 5ml/kg of water during the one hour of recovery, following which venous bloods, saliva samples, and capillary blood ketones will be collected.

Dietary intervention: Following each initial experimental trial, participants will be randomised to either; 1) maintain their habitual diet or; 2) low carbohydrate, ketogenic diet for 28 days. Day one will be considered as the day immediately post the experimental trial, however, all participants will be advised to commence their dietary allocation immediately following the experimental trial as the ability to restore depleted muscle glycogen will lead to rapid differentiation between the two trials. A detailed dietary education session (for either the habitual or ketogenic diet) will be provided following the pre-experimental test by a RD (i.e. two days prior to the exercise trial and commencement of the dietary allocation). The purpose of providing education two days prior to commencing the diet is to allow the participant time to organise their diet. Potential performance and health outcomes of each diet will not be discussed.

The ketogenic diet will contain <50g/day CHO, 15-20% protein and 75-80% fat, and will follow similar philosophies as previously reported. Participants in the ketogenic diet group will be provided with a nutrition plan developed by a RD that is specific to their taste preference, budget and preparatory skills as a guide. Total energy intake will be matched to the food diaries provided during screening, however, adjustments will be made during the study to prevent weight fluctuations. No medications or supplements known to affect immune function may be consumed during the entirety of the study. All participants will have daily access (phone and email) to a RD during the entirety of their participation.

Dietary monitoring: Participants will be required to provide an image-assisted weighed dietary record alongside a fudicial marker for two non-consecutive days between the days of 1 -7, 8 – 14 and 15 - 21 self-selected by the participant. Additionally, participants will record their intake for the 3 days prior and the initial 2 days during each testing block (i.e. days -5, -4, -3, -2 and -1, and 26, 27, 28, 29 and 30) using WhatsApp mobile phone app that can be viewed remotely to monitor compliance and provide immediate feedback. Each diet record will be coded using Food works Professional Edition, Version 8 (Xyris Software, Brisbane, Australia) by a two RDs and checked for accuracy. Participants will report their daily morning weight during each dietary condition (education will be provided for how to standardise weigh-ins). If there is a >2% difference in weight, participants will be required to undertake an immediate nutrition consult with an RD. Saliva samples will be collected in the morning, soon after waking and before breakfast, on days 7, 14 and 21. During the ketogenic diet condition, participants will be asked to self-measure their daily morning urine ketones (acetoacetate) using self-testing strips (Ketostix, Bayer) at a recommended time of 07:00 to 08:00 h and to measure capillary blood D-BHB at the same time on days 3, 7, 14 and 21, and to send a picture of the strips and ketone monitor to the primary researcher using WhatsApp for confirmation. Participants who do not comply with the study’s requirements or fail to exhibit positive daily urinary ketones after 7 days or for more than 85% of the days (i.e. 6 of 7 days) during days 8 - 31 will be requested to discontinue and excluded from the study. Urinary ketones will not be monitored during the control diet.

Training monitoring: Participants will self-report their morning resting HR and training data daily. Training load will be quantified via each session’s duration (h : min), average heart rate (HR), maximum HR (HRmax) and rate of perceived exertion (RPE). These variables will be used to calculate training impulse, using Banister TRIMP (see equation below) and accumulative session RPE.

TRIMP = D (change HR ratio)eb(change HR ratio)

where D = duration of training session, b = 1.92 (i.e. exponent for male athletes) and (change HR ratio) is determined using the following equation:

Change HR ratio = (HRex – HRrest)/(HRmax – HRrest),

where HRrest = the average heart rate during rest and HRex = the average HR during exercise.

Participants will be asked to replicate their training during the subsequent dietary condition to minimise the effect of training on physiological and performance outcomes.
Intervention code [1] 300795 0
Lifestyle
Comparator / control treatment
Participants will act as their own control (i.e. habitual diet trial). Additionally, following each dietary arm, participants will undergo a 14-day washout period. Participants will be asked to maintain or return to their habitual dietary and training habits. Participants will be required to report any illness, upper respiratory symptoms or gastrointestinal symptoms to the primary researcher. No supplements or medications that affect immune function may be consumed during this period. At the end of the washout period, participants will be required to complete the alternative dietary arm (i.e. habitual diet or ketogenic diet).
Control group
Active

Outcomes
Primary outcome [1] 305397 0
T-cell mediated immune response (i.e. peripheral blood T-cell subset count and balance, and stimulated cytokine production) using the Muse, RT-PCR and Magpix. T-cells will be stimulated using Infanrix-Hexa within a whole blood culture.
Timepoint [1] 305397 0
Immediately pre exercise, immediately post exercise and 1hr post exercise on days 0 and 31 of each dietary trial.
Primary outcome [2] 305398 0
Salivary immunoglobulin A concentration and secretion rate (i.e. amount of salivary immunoglobulin A collected divided by collection time). sIgA will be measured an using in-house ELISA method.
Timepoint [2] 305398 0
Immediatley pre exercise, immediately post exercise and 1hr post exercise on days 0 and 31 of each dietary trial. Additionally, on days 7, 14 and 21 of each dietary trial.
Primary outcome [3] 305399 0
Lymphocyte activation, determined by CD69+ expression on lymphocyte subpopulations (CD3+CD4+, CD3+CD8+, CD3-CD56+) using the flow cytometer.
Timepoint [3] 305399 0
Immediatley pre exercise, immediately post exercise and 1hr post exercise on days 0 and 31 of each dietary trial.
Secondary outcome [1] 345065 0
Time to exhaustion running at 70% VO2max. Simple regression equations will be used to estimate the speed required to elicit 70% VO2max from the VO2max test for the use in the experimental trial. Adjustments will be made to the running speed during the first 10 min based on real time measurements of oxygen uptake.
Timepoint [1] 345065 0
Assessed on days 0 and 31 of each dietary trial
Secondary outcome [2] 345066 0
Nutritional intake as determined by participants providing an image-assisted weighed dietary record alongside a fudicial marker for two non-consecutive days between the days of 1 -7, 8 – 14 and 15 - 21 self-selected by the participant. Additionally, participants will record their intake for the 3 days prior and the initial 2 days during each testing block (i.e. days -5, -4, -3, -2 and -1, and 26, 27, 28, 29 and 30) using WhatsApp mobile phone app that can be viewed remotely to monitor compliance and provide immediate feedback. Each diet record will be coded using Food works Professional Edition, Version 8 (Xyris Software, Brisbane, Australia) by a two registered dietitians and checked for accuracy.
Timepoint [2] 345066 0
Assessed on 2 non-consecutive days between the days 1 - 7, 8 - 14, 15 - 21, self selected by the participant, in addition to the 5 days prior to the follow up experimental trial. Each days nutritional intake will be coded and average nutrient intake will be determined for each dietary trial.

Eligibility
Key inclusion criteria
Endurance-trained male runners; VO2peak >50ml/kg/min; 20 - 40 years; history of competition; training for >2 years; run >50km/wk; habitually consuming a high carbohydrate diet; weight stable for >1 month.
Minimum age
20 Years
Maximum age
40 Years
Sex
Males
Can healthy volunteers participate?
Yes
Key exclusion criteria
Possess cardiovascular, metabolic, neurological, immunological or autoimmune disorders; baseline haematological values (leukocytes and erythrocyte counts) outside of normal range; experienced illness and/or upper respiratory symptoms within the previous 4 weeks; experienced severe gastrointestinal symptoms within the previous 4 weeks or has a history of gastrointestinal symptomology (e.g. irritable bowel syndrome); consumed medications or supplements known to effect immunity within previous 2 weeks; smoker or previously smoked; family member has died before the age of 50 due to a heart condition.

Study design
Purpose of the study
Prevention
Allocation to intervention
Randomised controlled trial
Procedure for enrolling a subject and allocating the treatment (allocation concealment procedures)
Each trial will be allocated a number (i.e. 1 = habitual diet and 2 = ketogenic diet). A set of 10 numbers will be generated using www.randomizer.org, which will infer initial dietary trial for each participant. If 5 of each (1 and 2) is not presented, this process will be completed until a counterbalanced randomisation occurs. Based on the order of participants recruitment, they will be allocated each number in consecutive order.
Methods used to generate the sequence in which subjects will be randomised (sequence generation)
Random number generation will be using www.randomizer.org
Masking / blinding
Open (masking not used)
Who is / are masked / blinded?



Intervention assignment
Crossover
Other design features
Nil
Phase
Not Applicable
Type of endpoint/s
Efficacy
Statistical methods / analysis
Null hypothesis testing and magnitude-based inferences will be used to describe the data.

Recruitment
Recruitment status
Completed
Date of first participant enrolment
Anticipated
28/05/2018
Actual
28/05/2018
Date of last participant enrolment
Anticipated
30/06/2018
Actual
30/06/2018
Date of last data collection
Anticipated
31/08/2018
Actual
31/08/2018
Sample size
Target
10
Accrual to date
Final
8
Recruitment outside Australia
Country [1] 10255 0
New Zealand
State/province [1] 10255 0
Auckland

Funding & Sponsors
Funding source category [1] 296291 0
University
Name [1] 296291 0
Auckland University of Technology
Country [1] 296291 0
New Zealand
Primary sponsor type
University
Name
Auckland University of Technology
Address
17 Antares Place, Second Floor, Rosedale, Auckland, New Zealand, 0632
Country
New Zealand
Secondary sponsor category [1] 295214 0
None
Name [1] 295214 0
Address [1] 295214 0
Country [1] 295214 0

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 297522 0
Auckland University of Technology Ethics Committee
Ethics committee address [1] 297522 0
Ethics committee country [1] 297522 0
New Zealand
Date submitted for ethics approval [1] 297522 0
Approval date [1] 297522 0
11/12/2017
Ethics approval number [1] 297522 0
17/410

Summary
Brief summary
Trial website
Trial related presentations / publications
Public notes

Contacts
Principal investigator
Name 74314 0
Mr David Shaw
Address 74314 0
17 Antares Place, Second Level, Sports Performance Research Institute of NZ (SPRINZ), Rosedale, Auckland, 0632
Country 74314 0
New Zealand
Phone 74314 0
+642108276137
Fax 74314 0
Email 74314 0
[email protected]
Contact person for public queries
Name 74315 0
David Shaw
Address 74315 0
17 Antares Place, Second Level, Sports Performance Research Institute of NZ (SPRINZ), Rosedale, Auckland, 0632
Country 74315 0
New Zealand
Phone 74315 0
+642108276137
Fax 74315 0
Email 74315 0
[email protected]
Contact person for scientific queries
Name 74316 0
David Shaw
Address 74316 0
17 Antares Place, Second Level, Sports Performance Research Institute of NZ (SPRINZ), Rosedale, Auckland, 0632
Country 74316 0
New Zealand
Phone 74316 0
+642108271637
Fax 74316 0
Email 74316 0
[email protected]

Data sharing statement
Will individual participant data (IPD) for this trial be available (including data dictionaries)?
No
No/undecided IPD sharing reason/comment
Individual participant data will only be made available to each participant.


What supporting documents are/will be available?

No Supporting Document Provided



Results publications and other study-related documents

Documents added manually
No documents have been uploaded by study researchers.

Documents added automatically
No additional documents have been identified.