Post-Exercise Protein Trial: Interactions between Diet and Exercise (PEPTIDE): study protocol for randomized controlled trial

Background Performing regular exercise is known to manifest a number of health benefits that mainly relate to cardiovascular and muscular adaptations to allow for greater oxygen extraction and utilization. There is increasing evidence that nutrient
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  STUDY PROTOCOL Open Access Post-Exercise Protein Trial: Interactions betweenDiet and Exercise (PEPTIDE): study protocol forrandomized controlled trial Abdullah F Alghannam 1* , Kostas Tsintzas 2 , Dylan Thompson 1 , James Bilzon 1 and James A Betts 1 Abstract Background:  Performing regular exercise is known to manifest a number of health benefits that mainly relate tocardiovascular and muscular adaptations to allow for greater oxygen extraction and utilization. There is increasingevidence that nutrient intake can affect the adaptive response to a single exercise bout, and that protein feeding isimportant to facilitate this process. Thus, the exercise-nutrient interaction may potentially lead to a greater responseto training. The role of post-exercise protein ingestion in enhancing the effects of running-based enduranceexercise training relative to energy-matched carbohydrate intervention remains to be established. Additionally, theinfluence of immediate versus overnight protein ingestion in mediating these training effects is currently unknown. The current protocol aims to establish whether post-exercise nutrient intake and timing would influencethe magnitude of improvements during a prescribed endurance training program. Methods/Design:  The project involves two phases with each involving two treatment arms applied in arandomized investigator-participant double-blind parallel group design. For each treatment, participants will berequired to undergo six weeks of running-based endurance training. Immediately post-exercise, participants will beprescribed solutions providing 0.4 grams per kilogram of body mass (g · kg − 1 ) of whey protein hydrolysate plus0.4 g · kg − 1 sucrose, relative to an isocaloric sucrose control (0.8 g · kg − 1 ; Phase I). In Phase II, identical proteinsupplements will be provided (0.4 + 0.4 g · kg − 1 · h − 1 of whey protein hydrolysate and sucrose, respectively), withthe timing of ingestion manipulated to compare immediate versus overnight recovery feedings. Anthropometric,expired gas, venous blood and muscle biopsy samples will be obtained at baseline and following the six-week training period. Discussion:  By investigating the role of nutrition in enhancing the effects of endurance exercise training, we willprovide novel insight regarding nutrient-exercise interactions and the potential to help and develop effectivemethods to maximize health or performance outcomes in response to regular exercise. Trial registration:  Current Controlled Trials registration number: ISRCTN27312291 (date assigned: 4 December2013). The first participant was randomized on 11 December 2013. Keywords:  Nutrition, Protein, Recovery, Aerobic training, Adaptation * Correspondence:  1 Human Physiology Research Group, Department for Health, University of Bath, Claverton Down, Bath BA2 7AY, UK Full list of author information is available at the end of the article TRIALS © 2014 Alghannam et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License (, which permits unrestricted use,distribution, and reproduction in any medium, provided the srcinal work is properly credited. The Creative Commons PublicDomain Dedication waiver ( ) applies to the data made available in thisarticle, unless otherwise stated. Alghannam  et al. Trials  2014,  15 :459  Background Performing regular exercise has a multitude of healthbenefits that stem from cardiovascular and skeletal muscleadaptations that occur in response to the exercise stimu-lus. Endurance exercise induces adaptations in the cardio- vascular system that allow an increase in capillary numberand plasma volume expansion, supporting a greater sur-face area for gas exchange and the movement of blood,and thus enhancing oxygen transport to the activemuscles [1]. Pronounced skeletal muscle adaptation to en-durance exercise include an increase in mitochondrialcontent and size (mitochondrial biogenesis) and increasedactivity of oxidative enzymes, which collectively improveoxidative capacity [2,3]. Accordingly, the capacity to per- form daily work tasks can be improved as a consequenceof endurance exercise.Adaptation to exercise is thought to occur as a conse-quence of the accumulated response of acute exercisebouts, while nutrient availability can influence the acuteresponse to exercise and may modulate chronic adapta-tions to exercise training [4]. Therefore, nutritional strat-egies provided in close temporal proximity to exercisehave the potential to improve training efficiency by en-hancing the magnitude of adaptations to the same train-ing stimulus [5]. Emerging acute mechanistic evidencesupports the potential benefit of post-exercise proteinfeeding in increasing muscle protein synthesis and miti-gating proteolysis associated with an endurance exercisebout [6-8]. Cycling-based endurance training studies in 2009 and 2011 have also proposed the role of protein in-gestion in supporting tolerance to intensified training inconcurrence with an increase in the magnitude of train-ing adaptations when repeated bouts of exercise areperformed [9-11]. Taken together, post-endurance exer- cise protein intake may provide means to facilitate aer-obic training adaptations. Nonetheless, the time courseeffects of prolonged post-exercise protein co-ingestion inenhancing the adaptive response to running-based endur-ance exercise training in adults remains to be established.Protein synthesis is an outcome of a complex physio-logical process by which external stimuli, such as contract-ile activity and nutrient availability, promotes phenotypiccharacteristics in skeletal muscle [4]. It is becoming clearthat both the exercise stimulus and protein feeding act in-dependently and synergistically in modulating muscle pro-tein synthesis, and consequently these subtle changes inmuscle quantity or quality can mediate worthwhile train-ing adaptations if sustained for weeks or months [4,12]. Furthermore, protein ingestion during and following anacute endurance exercise bout has been shown to increasemuscle protein synthesis and reduce muscle proteinbreakdown, and thus results in an increased whole body net protein balance [6,13]. In spite of the fact that endur- ance exercise does not typically result in muscle massaccrual, the changes in muscle protein synthesis followingendurance exercise are relevant to drive tissue repair andremodeling, in concurrence with the synthesis of non-contractile proteins, such as the mitochondria [14]. In-deed, acute and chronic endurance exercise has beendemonstrated to stimulate the mitochondrial protein syn-thetic response [15,16]. While the influence of protein in- gestion on mitochondrial biogenesis machinery is notclear at present [8,11,17], its synthesis appears to be stimulated by the availability of extracellular aminoacids in a dose-response manner [18]. This may inferthat exogenous protein availability can enhance the cap-acity for muscular adaptations, although the role of post-exercise protein ingestion in enhancing the effectsof running-based endurance exercise training remainslargely unknown [11,19]. Cardiovascular adaptations are a hallmark of endur-ance exercise training. An enhancement in oxidativecapacity and maximal oxygen uptake (V   O 2 max  ) in re-sponse to aerobic training with post-exercise protein in-gestion has been observed by some [10,11], but not all [20] studies. In concordance, the role of protein feedingin enhancing the adaptive response to endurance train-ing may not reside in the intramuscular milieu. Rather,noticeable cardiovascular improvements in V   O 2 max  viaan increase in plasma volume and plasma albumin con-tent have been shown with protein ingestion [10,11]. However, the addition of other nutrients such as caffeine,flavonoids, multivitamins and ribose [11,20], in addition to the absence of macronutrient-specific comparisons by notincluding a carbohydrate-only supplement [10], makes itdifficult to conclude whether these improvements in train-ing adaptation are a result of protein intake  per se . Inter-estingly, the magnitude of improvement in V   O 2 max  duringendurance training was greater when whey protein wasingested relative to a placebo [10], or when milk-basedprotein available in the form of chocolate milk was co-ingested, when compared to an energy-matched carbohy-drate supplement and a placebo [11]. Collectively, thesefindings may infer nutrient-specific effects of post-exercisesupplementation on endurance training adaptations.In summary, recent scientific evidence from acute la-boratory investigations and relatively extended cycling-based training studies supports the notion that nutrientintake can increase protein accretion and, ultimately,this may influence the magnitude of the training effect.This nutrient-exercise interaction may modulate the adap-tive response to training and protein feeding appears to bean important factor in mediating this process. However,examining the long-term outcomes of these acute mechan-istic models, particularly during free living endurance-typeexercise, require further investigation. This project aims toaddress two issues (Phase I and Phase II). Phase I willaddress whether protein co-ingestion with carbohydrate Alghannam  et al. Trials  2014,  15 :459 Page 2 of 12  immediately following exercise can increase the magnitudeof cardiovascular and intramuscular training adaptations toendurance training when compared with ingestion of carbohydrate alone. Phase II while address to what extentthe timing of protein co-ingestion (overnight versus imme-diate feeding) impacts upon those training adaptations.It is hypothesized that the inclusion of protein in apost-exercise nutritional supplement will increase themagnitude of improvement in cardiovascular, but notintramuscular, adaptations following a long-term endur-ance training intervention in Phase I. Furthermore, it ishypothesized that overnight protein feeding will enhancethe intramuscular adaptive response (markers of mito-chondrial biogenesis) to endurance training relative toimmediate feeding in Phase II. Methods/Design Approach to research questions The primary outcomes of this trial are the assessment of cardiovascular (changes in V   O 2 max , plasma albumin con-tent and plasma volume) and intramuscular (selectedgenes and proteins involved in cellular adaptive pro-cesses) training-induced adaptations, and whether pro-tein co-ingestion (immediate or overnight) improves themagnitude of these adaptations. In accordance, two phasesof testing will be conducted.Phase I will be addressed by evaluating a carbohydrate-protein mixture against an energy-matched carbohydratecontrol to examine the influence of post-exercise proteinintake in enhancing endurance training adaptations. Par-ticipants in Phase I will be randomly allocated to a groupreceiving an isocaloric carbohydrate supplement or agroup receiving a carbohydrate-protein supplement, in arandomized investigator-participant double-blind manner,whereby the chief investigator (AFA) and participants willbe blinded from the trial allocation. The acute post-exercise phase is important when considering the meta-bolic priority for recovery to be initiated [3,21] and the po- tentiated sensitivity to nutritional interventions duringthis period [22]. It has been established that glycogenresynthesis and amino acid uptake rates are augmentedfollowing an exercise bout, and that the ingestion of carbohydrate and protein stimulate this process [6,23-25]. This has subsequently prompted large numbers of studiesto investigate the role of combined carbohydrate-proteiningestion in restoring glycogen stores, potential attenu-ation of decrements of skeletal muscle functional integrity and subsequent exercise performance [12,26]. It is now  generally accepted that incorporating protein into a post-exercise feeding regimen is a viable strategy to enhancerecovery, mainly through observations in acute basedstudies [7,8,21,27-33]. Yet, the effects of protein ingestion are more likely to be realized beyond the acute recovery phase, given that upregulation of endurance exercise-specific gene expression peaks between 10 and 24 hoursfollowing an exercise bout, and may surpass 96 hoursfor muscular recovery and adaptive remodeling to occur,suggesting that repeated bouts of exercise are requiredto detect worthwhile training adaptations [3,34]. Given that the target population will be physically un-trained and subjected to running-based aerobic exercisetraining, and that reversal of these training-induced ad-aptations are relatively slow in such individuals [35], thestudy will adopt a randomized parallel group design ineach phase with a relatively larger cohort of participantsto account for inter-individual variability. The durationof the training period will be six weeks. This durationwas deemed adequate based on previous literature, whichdemonstrated that approximately four weeks (total num-ber of sessions was between 20 and 22 during the entireprotocol) elicits changes in V   O 2 max  of 6 to 14%, oxidativeenzyme activities of 35 to 50% and mitochondrial proteincontent of 50 to 100% in untrained and moderately trained individuals [11,36,37]. Phase II of testing will examine the influence of pro-tein timing upon mediating the adaptive response to en-durance exercise. Participants will be randomly assignedto a group receiving the same carbohydrate-proteinmixture in Phase I, but with one group ingesting thesupplement immediately post-exercise, while the secondgroup supplementation will take place during overnightrecovery in a double-blinded manner. The interactionbetween exercise and protein ingestion on mitochon-drial protein synthesis may lie outside the acute recov-ery phase (over eight hours post-exercise) and undercircumstances of rested mitochondrial turnover rates[8,19]. Moreover, the provision of dietary protein prior to sleep was shown to be an effective nutritional strategy tofurther augment muscle protein synthesis during over-night recovery [38]. Coupled with the fact that exogenousamino acids stimulate mitochondrial synthetic response inthe rested state [18], protein intake during overnight re-covery may increase the adaptive response to endurancetraining relative to immediate feeding. Therefore, compar-isons between immediate versus overnight protein inges-tion (Phase II) are warranted to provide an insight into themolecular and transcriptomic responses that could extendthe knowledge regarding the relationship of nutrient-exercise stimuli to mitochondria biogenesis.It has also been established that rapid hyperaminoacide-mia (particularly leucine) is required to maximize muscleprotein synthetic response; however, this response isreturned to baseline approximately three hours after a sin-gle bolus of ingested protein within the recommendedquantities (approximately 0.3 to 0.4 grams per kilogram of body mass; g·kg BM − 1 ) for maximal muscle protein syn-thesis [39-41]. Therefore, it may be postulated that a Alghannam  et al. Trials  2014,  15 :459 Page 3 of 12  second bolus would be advantageous in inducing hypera-minoacidemia and would supply sufficient amounts of leucine into circulation to prompt the  ‘ leucine trigger ’ [40,42,43], consequently maximizing muscle protein syn- thesis during post-exercise overnight recovery. Participants A total of 32 research participants will be recruited foreach phase from the local community via public advertise-ment (including online posts and distribution of flyers)and personal communication. Upon contacting the chief investigator (AFA), participants will be provided with aparticipant information sheet detailing the study aims andrequirements. Participants who express their interest willbe scheduled for an initial meeting to assess their eligibil-ity and provide further details regarding the study require-ments. An informed consent form will be obtained fromindividuals who agree to participate following the briefing,indicating their full understanding of the study and theirprotected rights for confidentiality and withdrawal fromthe study. Thereafter, a medical health questionnaire willbe undertaken by each participant to ensure the absenceof any physical, hematologic, metabolic or any otherhealth conditions. If any of the factors above are present,the volunteer will be deemed unfit to participate in thestudy and will consequently be excluded from taking part.Further health screening and a patient-specific directionfor the use of anesthetic will be completed and sent to amedical practitioner, who will respond to confirm theabsence of any contraindications prior to any localanesthesia administration.The protocol described herein was reviewed and ap-proved by the National Health Service (NHS) South West3 Research Ethics Committee (approval number: 13/SW/0239). The project was subsequently registered as a con-trolled trial (Current Controlled Trials registration num-ber: ISRCTN27312291). The individuals who participatein Phase I of testing will not be eligible to take part inPhase II. Inclusion criteria The inclusion criteria for this study are as follows: 1. Both male and female volunteers.2. Aged between 18 and 48 years.3. Individuals free from cardiovascular, metabolic or joint disease as determined by a standard healthquestionnaire.4. No engagement in structured physical activity for morethan two hours per week during the last two years.5. Nonsmoker. Exclusion criteria The exclusion criteria for this study are as follows: 1. Known or suspected food intolerances, allergies orhypersensitivity.2. Any bleeding disorder or taking medication whichimpacts blood coagulation.3. Known tendency towards keloid scarring.4. Known sensitivity or allergy to any local anestheticmedicines.5. Any reported use of substances which may poseundue personal risk to participants or introduce biasinto the experiment.6. Any other condition or behavior deemed either topose undue personal risk to participants orintroduce bias into the experiment. The sex of participants will be included in a stratifiedrandomization scheme using a computer-based randomnumber generator produced by an academic supervisor(JAB), who is not responsible for trial enrollment, nutri-tional preparation or provision. The only interaction by JAB with participants will be for obtaining muscle biop-sies at baseline and follow-up, and thus he will be un-aware of the assigned coding numbers to participants,rendering him unable to determine trial allocation toany participant. Details of the overall randomizationscheme will only to be published once follow-up mea-surements are completed in order to complicate deci-phering of the allocation sequence by those involved intrial enrollment [44]. Experimental design The proposed project involves two distinct phases of testing, each contrasting two nutritional interventions ina randomized investigator-participant double-blind par-allel group design. The independent variable will be theprecise nutritional intervention ingested following exer-cise in Phase I, while timing of ingestion will be theindependent variable in Phase II. Specifically, those par-ticipants recruited for Phase I (n =32, approximately) of testing will receive a supplement containing carbohy-drate (sucrose) plus protein (whey protein hydrolysate)at an ingestion rate of 0.4 and 0.4 g · kg BM − 1 respect-ively. The second group will be a control group,whereby an ingestion of an isocaloric carbohydrate sup-plements will be assigned (0.8 g · kg BM − 1 ). The two nu-tritional interventions provided in Phase II (n =32,approximately) will be identical (0.4 g · kg BM − 1 carbo-hydrate +0.4 g · kg BM − 1 whey protein hydrolysate),while the timing of intake will be manipulated. A groupwill ingest the carbohydrate-protein supplement imme-diately post-exercise and one hour later, while the over-night recovery group will receive the supplements onehour prior to sleep, and at 02:00, as an overnight nutri-tional intervention. The nutritional information for thesupplements is provided in Table 1. Alghannam  et al. Trials  2014,  15 :459 Page 4 of 12  Supplements will be provided in a sachet form alongwith shaker bottles with measurement scales. Solutionpreparation will be instructed to participants to achievea volume of ingestion of 10 ml ·kg BM − 1 . The entireprotocol will be eight weeks duration and will consistof: (i) a baseline testing week with two exercise sessions,(ii) the first, second and third weeks of training at70%   VO 2 max , (iii) the fourth, fifth and sixth weeks of train-ing at 75% V   O 2 max  and (iv) a final week for follow-upmeasurements. A total of 26 endurance-based trainingsessions will be prescribed for each participant. Baseline testing Prior to the start of the training period, participants arerequired to attend the laboratory on two occasions. Thefirst visit will include anthropometric assessment of height,weight and body mass. Participants will also undergo anassessment of their running economy (V   O 2  · km − 1 ) andV   O 2 max  (details provided below). The second prelimin-ary visit will then be arranged within a week followingthe first laboratory visit. A 48-hour standardization of life-style will be employed (discussed below). This visit will in- volve obtaining fat mass percentage using bioelectricalimpedance analysis (BIA), followed by a resting expiredgas sample to measure resting metabolic rate (RMR).After laying on a semi-supine bed for five minutes, twofive-minute baseline resting expired gas samples will betaken for the estimation of RMR, along with resting heartrate via short-range telemetry (Polar FT2, Kempele,Finland). A venous blood sample (10 ml) will also beobtained from an antecubital vein to measure differentplasma metabolites, in addition to hematological parame-ters for the estimation of plasma volume change. Subse-quently, an 80 to 100 mg muscle biopsy sample will beobtained from the vastus lateralis under a local anesthetic(1% lidocaine; Hameln Pharmaceuticals Ltd., Brockworth,United Kingdom). In relation to eumenorrheic female par-ticipants, all measurements will be conducted three to10 days after the onset of menses (follicular phase) to en-sure low levels of circulating female hormones and there-fore minimize any measurement errors associated withmenstrual cycle [45]. Two exercise sessions will then beemployed during the baseline testing week that requireparticipants to run on a motorized treadmill for 30 mi-nutes per day. These were aimed to allow participants togauge the relative intensity required during their pre-scribed training intervention, while also familiarizing themwith treadmill running, before the commencement of thesix weeks training intervention. Training intervention Exercise sessions performed in the baseline testing weekwill last for 30 minutes and thereafter the duration of sessions will progressively increase to 40, 50 and 60 mi-nutes in week one, weeks two to three, and weeks fourto six, respectively. Prior to any exercise session, a five-minute warm-up at 60% V   O 2 max  will be superimposed,followed by treadmill running at a speed correspondingto 70% V   O 2 max  for the durations indicated above. Thetarget % V   O 2 max  will be attained by providing partici-pants with their relative speeds at any given intensity during the training intervention, which will be determinedby linear regression (Excel 2010, Microsoft, Redmond,Washington, United States) during the first baseline visit.During the midpoint (training week three), the speed willbe increased to elicit 75% V   O 2 max . Only water consump-tion will be permitted during exercise sessions, which willbe consumed  ad libitum . Upon cessation of each exercisesession in Phase I, a post-exercise nutritional supplementwill be ingested immediately, while a second bolus willthen be consumed one hour after the end of exercise. Inrelation to Phase II of testing, one group will ingest apost-exercise nutritional supplement in a similar mannerto Phase I, while the other group will ingest an identicalsupplement one hour prior to sleep and at 02:00. Duringthe nutritional provision periods, participants will beinstructed to consume only water to avoid any confound-ing results relating the outcome variables of the study.Once every fortnight, participants will report to the la-boratory to provide the nutritional supplements for thesubsequent two-week training block, with a total of three scheduled meetings throughout the training inter- vention. This visit is also to confirm the adherence of participants to the prescribed training and supplemen-tation procedures.Although several risk factors will be screened beforeparticipants take part in exercise, the prescribed physical Table 1 Nutritional information of the supplementsprovided in Phase I and Phase II of the trial High sucrose Sucrose+wheyprotein hydrolysateSucrose (g/l)  80 40 Lactose (g/l)  -  ≤ 2.3* Protein (g/l)  - 40 Fat (g/l)  -  ≤ 2.4* Sodium (g/l)  - 0.72 Potassium (g/l)  - 0.10 Calcium (g/l)  - 0.15 Magnesium (g/l)  - 0.01 Phosphorous (g/l)  - 0.19 Chloride (g/l)  - 0.29 Energy (kcal/)  320 320 *Assay unable to detect values below this number. The caloric content for fatand lactose was therefore assumed to be negligible. Alghannam  et al. Trials  2014,  15 :459 Page 5 of 12
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