NAP: Recovery (2014)


Nelson AR, Phillips SM, Stellingwerff T, Rezzi S, Bruce SJ, Breton I, Thorimbert A, Guy PA, Clarke J, Broadbent S, Rowlands DS. A protein-leucine supplement increases branched-chain amino acid and nitrogen turnover but not performance. Med Sci Sports Exerc. 2012; 44 (1): 57-68

PubMed ID: 21685813
Study Design:
Randomized Crossover Trial
A - Click here for explanation of classification scheme.
Quality Rating:
Positive POSITIVE: See Quality Criteria Checklist below.
Research Purpose:

To determine the effect of post-exercise protein-leucine coingestion with CHO-lipid on subsequent high-intensity endurance performance and to investigate candidate mechanisms using stable isotope methods and metabolomics.

Inclusion Criteria:
Well-trained male cyclists.
Exclusion Criteria:
  • Failed to pass a health screening
  • Recently donated blood
  • Recently consumed caffeine, alcohol, medications or drugs
  • Smoked during the experimentally controlled period.
Description of Study Protocol:


Not discussed.


  • This is a double-blind, randomized crossover study during a six-day block of controlled high-intensity training and diet
  • Before the first experimental block, participants recorded their habitual training and diet for seven and three days, respectively. Training was tapered in the three days before the experimental block, including a rest day before starting.
  • To standardize lead-in fatigue and minimize diet variation, this training and dietary regimen was repeated preceding Block Two
  • Each block comprised four intermittent high-intensity rides
    • Day One (three hours of cycling) consisted of a warm-up (15 minuted at 30%, 10 minutes at 40% and six minutes at 50% Wmax), followed by loading intervals: Three blocks of 10  two-minute intervals at 90/80/70% Wmax (two minutes at 50% Wmax between intervals and six minutes at 50% Wmax between blocks) and a cool down of 11 minutes at 30% Wmax.
    • Day Two (2.5 hours of cycling) comprised a warm-up (10 minutes at 30%, 10 minutes at 40%, five minutes at 50% Wmax), followed by two blocks of longer loading intervals (four five-minute intervals at 70% Wmax interspersed with three of five minutes at 50% Wmax and three of four minutes at 70%, interspersed with three of four minutes at 50% Wmax) and separated by three of one minute at 90% and three of one minute at 80% Wmax (interspersed by two minutes at 50% Wmax)
    • Days Four and Six comprised 90 minutes at 50% of Wmax followed by the repeated sprint performance test
    • Rides on Days Three and Five comprised 60 minutes at 30% of Wmax.
  • All rides were conducted at the same time of day for a given participant.

Blinding Used


  • Isocaloric supplements fed one to three hours after exercise during a six-day training block (intense intervals, recovery, repeated-sprint performance rides). Daily protein intake was clamped at 1.9g per kg-1 per day-1 (LEUPRO) and 1.5g per kg-1 per day-1 (control)
  • LEUPRO: Leucine/protein/CHO/fat supplement (7.5g/20g/89g/22g per hour-1)
  • Control: CHO/fat (119g/22g per hour-1).

Statistical Analysis

  • The effect of treatment on outcomes was estimated with mixed modeling. Most outcome variables were 100x log-transformed before modeling to reduce non-uniformity of error and to express outcomes as percentages, with the exception of data sets with negative values (nitrogen balance).
  • Most outcomes and comparisons were generated from fixed-effects models based on the interaction between the respective levels of treatment, test day and order of treatment
  • For the analysis of sprint mean power, sprint number was a numeric effect (as in linear regression)
  • Appropriate random-effect models for each parameter included all or some of between-athlete variation, additional treatment associated variation and additional variation associated with moving between test days
  • Variability between blocks at baseline in blood measures was identified a priori as a potential confounder; value at baseline was hence included as a covariate within the appropriate model
  • All covariates were first normalized to and expressed as a proportion of the within-subject SD for the covariate
  • For performance, we used 0.93% (0.3x3.1%) as the threshold and for a mechanistic outcome, we used the standardized difference (effect size, ES).
Data Collection Summary:

Stable isotope infusions [1-(13)C-leucine and 6,6-(2)H2-glucose], mass spectrometry-based metabolomics and nitrogen balance methods were used to determine the effects of LEUPRO on whole-body branched-chain amino acid (BCAA) and glucose metabolism and protein turnover.

Timing of Measurements

  • Whole-body leucine turnover was measured using a primed constant 1-13C-leucine infusion during the three-hour post-exercise recovery period on Day One and at rest (pre-exercise) and during steady-state exercise on Day Six
  • Expired breath samples for indirect calorimetry were directed through a five-L mixing chamber attached to a Douglas bag for 10- to 12-minute gas collections at rest and three to five minutes during exercise
  • Blood samples for additional parameters were collected on Days One and Six at sampling times, as described for leucine/glucose infusions, as well as after exercise on Day Six. Pre-exercise and post-exercise samples were taken on Days Two and Four by venipuncture.
  • Throughout the six-day block, 24-hour urine was collected for quantification of nitrogen excretion and urinary metabolite analysis
  • Sweat was collected during exercise on Day Two and during the 90-minute steady-state ride on Day Six
  • Net nitrogen balance was calculated during five approximately 24-hour collections (Day One being only approximately 12 hours) to a total of 108 hours
  • Measured nitrogen outputs were urinary urea and creatinine, with additional estimated nitrogen losses from sweat at rest and during exercise on Days Two and Six.

Dependent Variables

  • Performance
  • Plasma essential and total amino acid concentration
  • Plasma and urinary concentrations of substrates and metabolites relating to the branched-chain amino acids, the urea cycle, the metabolism of alanine and aspartate, the degradation of lysine or the metabolism of arginine and proline
  • Plasma concentrations of glucose and insulin
  • Metabolomics
  • CK concentration
  • Leucine turnover
  • Nitrogen balance
  • Amino acid metabolomics
  • RER
  • Whole-body leucine and glucose kinetics. Whole-body leucine breakdown, non-oxidative disposal, oxidation and balance immediately after exercise (recovery, zero minutes) and during recovery (60 minutes to 180 minutes) on Day One and at rest and during steady-state exercise on Day Six.
Independent Variables

A leucine/protein/CHO/fat supplement (LEUPRO 7.5g/20g/89g/22g per hour-1, respectively) or isocaloric CHO/fat control (119g/22g per hour-1).

Control Variables
  • Standardized diet and training regimen prior to testing
  • Controlled diet during experimental blocks (all food provided).
Description of Actual Data Sample:
  • Initial N: 12 males
  • Attrition (final N): 12
  • Age (mean±SD): 35±10 years
  • Ethnicity: NA
Other Relevant Demographics
  • Maximal oxygen uptake (VO2max): 64.8±6.8ml per kg-1
  • Peak power output (Wmax): 355±36 W
  • Cyclists had 9±4 years of training history, with a recent weekly training volume of 10±1 hours.
  • Height: 182±5cm
  • Body mass: 76.9±6.5kg
  • Location: New Zealand.
Summary of Results:

Key Findings

  • After exercise, LEUPRO increased BCAA levels in plasma (2.6-fold; 90% confidence limits, ×/÷1.1) and urine (2.8-fold; ×/÷1.2) and increased products of BCAA metabolism plasma acylcarnitine C5 (3.0-fold; ×/÷0.9) and urinary leucine (3.6-fold; ×/÷1.3) and beta-aminoisobutyrate (3.4-fold; ×/÷1.4), indicating that ingesting approximately 10g leucine per hour during recovery exceeds the capacity to metabolize BCAA
  • LEUPRO increased leucine oxidation (5.6-fold; ×/÷1.1) and non-oxidative disposal (4.8-fold; ×/÷1.1) and left leucine balance positive relative to control
  • With the exception of Day One (LEUPRO, 17±20mg N per kg-1; control, -90±44mg N per kg-1), subsequent (Days Two to Five) nitrogen balance was positive for both conditions (LEUPRO, 130±110mg N per kg-1, control, 111±86mg N per kg-1)
  • Compared with control feeding, LEUPRO lowered the serum creatine kinase concentration by 21% to 25% (90% confidence limits, ±14%), but the effect on sprint power was trivial (Day Four, 0.4±1.0%; Day Six, -0.3±1.0%)
  • There were no clear effects of treatment on the RER (range of means, 0.86 to 0.90) during recovery from exercise on Day One and at rest or during exercise on Days Four and Six
  • Overall mean plasma glucose and insulin concentrations during the three-hour recovery period after exercise on Day One in the control were 6.9±1.6mmol per L-1 and 89±118mcIU per ml-1; the LEUPRO supplement led to a 12% reduction (±7%; ES, -0.80±0.51; P=7e-3) and a possible increase of 17% (±34%; ES, 19±34, P=0.23), respectively
  • Nitrogen balance was positive on all of the five 24-hour collection periods with the LEUPRO supplement; with control, nitrogen balance was negative from the completion of exercise on Day One through to the morning of Day Two
  • On Day One in the control, the total plasma amino acid concentration declined with time from the start of recovery
  • LEUPRO supplementation led to an extremely large increase in plasma leucine (3.5-fold) and a very large increase in essential (2.2-fold) and total amino acid (1.7-fold) concentrations, relative to the control
  • LEUPRO ingestion during recovery increased the concentration of plasma and urinary metabolic intermediates of BCAA degradation, indicative of protein and leucine intake that exceeded the whole-body capacity to metabolize BCAA
  • LEUPRO led to small to moderate reductions of plasma CK concentrations before and after exercise on Days Four and Six.
Author Conclusion:
  • Post-exercise supplementation with LEUPRO resulted in a positive whole-body net leucine balance, reduced plasma CK and led to the accumulation of plasma and urinary amino acids and metabolites during the recovery period and resulted in positive leucine and nitrogen balance during the immediate hours after an intense endurance exercise
  • However, the supplement provided no clear benefit to subsequent performance.
Funding Source:
Nestec Ltd., Vevey,Switzerland
Pharmaceutical/Dietary Supplement Company:
Reviewer Comments:
  • Results are interesting, however it is a small sample size
  • Further studies on large sample, different ethnic groups and increase in duration of the study is required.
Quality Criteria Checklist: Primary Research
Relevance Questions
  1. Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? (Not Applicable for some epidemiological studies) Yes
  2. Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about? Yes
  3. Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dieteticspractice? Yes
  4. Is the intervention or procedure feasible? (NA for some epidemiological studies) Yes
Validity Questions
1. Was the research question clearly stated? Yes
  1.1. Was (were) the specific intervention(s) or procedure(s) [independent variable(s)] identified? Yes
  1.2. Was (were) the outcome(s) [dependent variable(s)] clearly indicated? Yes
  1.3. Were the target population and setting specified? Yes
2. Was the selection of study subjects/patients free from bias? Yes
  2.1. Were inclusion/exclusion criteria specified (e.g., risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study? Yes
  2.2. Were criteria applied equally to all study groups? N/A
  2.3. Were health, demographics, and other characteristics of subjects described? Yes
  2.4. Were the subjects/patients a representative sample of the relevant population? ???
3. Were study groups comparable? N/A
  3.1. Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT) Yes
  3.2. Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline? N/A
  3.3. Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.) N/A
  3.4. If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis? N/A
  3.5. If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.) N/A
  3.6. If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")? N/A
4. Was method of handling withdrawals described? N/A
  4.1. Were follow-up methods described and the same for all groups? N/A
  4.2. Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate) and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%.) N/A
  4.3. Were all enrolled subjects/patients (in the original sample) accounted for? N/A
  4.4. Were reasons for withdrawals similar across groups? N/A
  4.5. If diagnostic test, was decision to perform reference test not dependent on results of test under study? N/A
5. Was blinding used to prevent introduction of bias? Yes
  5.1. In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate? Yes
  5.2. Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.) N/A
  5.3. In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded? N/A
  5.4. In case control study, was case definition explicit and case ascertainment not influenced by exposure status? N/A
  5.5. In diagnostic study, were test results blinded to patient history and other test results? N/A
6. Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described? Yes
  6.1. In RCT or other intervention trial, were protocols described for all regimens studied? Yes
  6.2. In observational study, were interventions, study settings, and clinicians/provider described? N/A
  6.3. Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect? Yes
  6.4. Was the amount of exposure and, if relevant, subject/patient compliance measured? Yes
  6.5. Were co-interventions (e.g., ancillary treatments, other therapies) described? N/A
  6.6. Were extra or unplanned treatments described? N/A
  6.7. Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups? N/A
  6.8. In diagnostic study, were details of test administration and replication sufficient? N/A
7. Were outcomes clearly defined and the measurements valid and reliable? Yes
  7.1. Were primary and secondary endpoints described and relevant to the question? Yes
  7.2. Were nutrition measures appropriate to question and outcomes of concern? Yes
  7.3. Was the period of follow-up long enough for important outcome(s) to occur? Yes
  7.4. Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures? Yes
  7.5. Was the measurement of effect at an appropriate level of precision? Yes
  7.6. Were other factors accounted for (measured) that could affect outcomes? Yes
  7.7. Were the measurements conducted consistently across groups? N/A
8. Was the statistical analysis appropriate for the study design and type of outcome indicators? Yes
  8.1. Were statistical analyses adequately described and the results reported appropriately? Yes
  8.2. Were correct statistical tests used and assumptions of test not violated? Yes
  8.3. Were statistics reported with levels of significance and/or confidence intervals? Yes
  8.4. Was "intent to treat" analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a dose-response analysis)? N/A
  8.5. Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)? N/A
  8.6. Was clinical significance as well as statistical significance reported? Yes
  8.7. If negative findings, was a power calculation reported to address type 2 error? N/A
9. Are conclusions supported by results with biases and limitations taken into consideration? Yes
  9.1. Is there a discussion of findings? Yes
  9.2. Are biases and study limitations identified and discussed? Yes
10. Is bias due to study's funding or sponsorship unlikely? Yes
  10.1. Were sources of funding and investigators' affiliations described? Yes
  10.2. Was the study free from apparent conflict of interest? Yes