The effect of a 4-week ketogenic diet on immune function in endurance athletes.

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.