NAP: Recovery (2014)


Ferguson-Stegall L, McCleave EL, Ding Z, Doerner III PG, Wang B, Liao YH, Kammer L, Liu Y, Hwang J, Dessard BM, Ivy JL. Postexercise carbohydrate–protein supplementation improves subsequent exercise performance and intracellular signaling for protein synthesis. J Strength Cond Res. 2011: 25(5): 1,210–1,224.



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 investigate the effects of dairy-based carbohydrate (CHO) and protein (PRO) recovery supplement (chocolate milk) on post-exercise muscle glycogen synthesis, activation of key signaling PROs involved in PRO synthesis [mammalian target of rapamycin phosphorylation (mTOR), rpS6, and elF2Be] and degradation (FOXO3A and ubiquitination), biochemical measures of muscle damage and inflammation and subsequent aerobic endurance performance.

Inclusion Criteria:

Trained cyclists and triathletes who regularly ride distances of 60 miles to 100 miles (3.5 hours to six hours).


Exclusion Criteria:

Not clearly defined.

Description of Study Protocol:

Randomized, double-blinded crossover study. Ten cyclists performed three trials, cycling 1.5 hours at 70% maximal oxygen consumption (VO2max) at 10-minute intervals. The cyclists then had a four-hour recovery period where they received the supplement intervention and had muscle biopsies performed. After the recovery period the cyclists completed a 40km time trial (TT).

Blinding Used

Double-blinded trial.


  • In three separate crossover exercise trials, subjects were given the following supplements immediately after the 1.5-hour ride and at two hours recovery: 
    • A CHO and PRO supplement in the form of organic, low-fat chocolate milk (11.48g CHO, 3.67g PRO, 2.05g fat, 79.05kcal, 3.12g CHO: 1g PRO per 100ml)
    • An isocaloric CHO supplement (15.15g CHO, 0g PRO, 2.05g fat, 79.05kcal per 100ml)
    • A placebo supplement (0g CHO, 0g PRO, 0g fat, 0kcal per 100ml).
  • Subjects were instructed to drink the supplement provided within five minutes. The amount of supplement provided was stratified according to body weight ranges. Subjects weighing less than 140 lbs got 500ml per supplement, 395.25kcal each, totaling 1,000ml and 790.5kcal during the recovery period. Subjects weighing 140 to 170 lbs got 600ml per supplement, 474.3kcal each, totaling 1,200ml and 948.6kcal during the recovery period. Subjects weighing more than 170 lbs got 700ml per supplement, 553.35kcal, totaling 1,400ml and 1,106.7kcal during the recovery period.

Statistical Analysis

  • Two-way ANOVA was used to analyze plasma insulin, glucose, lactate, myoglobin, CPK, FFA, glycerol, cortisol and cytokines
  • One-way ANOVA was used to analyze time trial measures (TT time, average power output, average heart rate, average RPE)
  • Two-way ANOVA was used to analyze differences in muscle glycogen re-synthesis 
  • Two-way ANOVA was used to analyze signaling PRO phosphorylation or content
  • Two-way ANOVA was used to analyze substrate oxidation and energy expenditure during recovery period 
  • Post-hoc analysis was performed when significance was found using the least significant difference.
Data Collection Summary:

Timing of Measurements

  • Before the start of their experimental trials, subjects came to the lab for maximal oxygen consumption and maximal workload determination, and maximum power output in Watts was calculated. Three to five days after the maximal oxygen consumption test, subjects came to the lab after a 12-hour overnight fast for a familiarization session. Subjects did a 40-minute portion of the glycogen depletion ride, including intervals at the end, on the same ergometer used for the maximal oxygen consumption test. After this ride subjects rested for one hour, then performed the 40km cycling time trial. There were no blood or muscle samples collected during the familiarization session.
  • For the experimental trial, subjects came to the lab after an overnight 12-hour fast. Resting heart rate was recorded using a monitor and a resting blood sample was drawn via a catheter inserted into a forearm vein. Heart rate was recorded at the beginning of each exercise bout, every 30 minutes during exercise and every 30 minutes during the recovery phase. Rating of perceived exertion (RPE) on a Borg scale were obtained during exercise at the same time points as heart rate.
  • Following the glycogen depletion ride muscle biopsies were done immediately, at 45 minutes into recovery and at four hours after the start of recovery. Blood collections were taken at the same times as the biopsies and at two hours into the recovery period. Respiratory exchange ratio (RER) was measured at four different times during the recovery time while subjects rested quietly for 10 minutes. During the TT following the recovery period, blood samples were collected at the beginning of the TT and at minutes 30 and 60 and at the completion of the TT. Each of the three experimental trials was separated by at least seven days but no more than 14 days.

Dependent Variables

  • Muscle glycogen resynthesis during recovery
  • Indicators of muscle damage and inflammation:
    • Myoglobin
    • Creatinine phosphokinase and cytokines interleukin-6 [IL-6], IL-8, IL-10, IL-1 receptor antagonist and TNFa.
  • Substrate use
  • Responses of insulin, glucose, lactate, free fatty acids, glycerol and cortisol to the different treatments
  • Phosphorylation of PROs controlling PRO synthesis and degradation and post-recovery TT performance.

Independent Variables

 In separate trials, subjects were given:

  • A CHO and PRO supplement in the form of organic, low fat chocolate milk
  • An isocaloric CHO supplement
  • A placebo.

Control Variables

  • Subjects were given 250ml of water at 15 minute intervals throughout the exercise trial and ad libitum during the recovery period
  • Fans were used to cool subjects to limit thermal stress during the exercise trial
  • The same investigator verbally encouraged the same subject during all three experimental trials. 


Description of Actual Data Sample:
  • Initial N: Ten cyclists (five males, five females
  • Attrition (final N): No attrition reported.
  • Age: The mean age was 31.8±.6 years (32.2±2.4 years for males, 31.4±2.5 years for females)
  • Other relevant demographics: All subjects were trained cyclists and triathletes who regularly ride distances of 60 to 100 miles (3.5 to six hours). The mean number of years training and racing was 5.0±1.1 years.


  • Mean mass of subjects: 67.8±2.6kg (72.5±3.1kg males, 63.1±3.2kg females)
  • Mean height: 171.1±3.4cm (176.8±3.1cm males, 165.4±5.1cm females)
  • Mean VO2max (L per minute-1): 3.6±0.2 (4.2±0.2 males, 3.0±0.1 females)
  • Mean VO2max (ml per kg-1 per minute-1): 52.6±2.3 (57.7±2.8 males, 47.6±1.5 females).


Austin, Texas.

Summary of Results:

Key Findings

  • The time trial time was significantly shorter in CM compared to in both CHO and PLA (79.43±2.11 vs. 85.74±3.44 and 86.92±3.28, P≤0.05)
  • Average power output (Watts) was significantly higher in CM compared to in both CHO and PLA (P≤0.05)
  • Heart rate during the TT was significantly higher in CM than in CHO or PLA (162.8±5.6 vs. 145.9±7.4 and 148.9±7.1, P≤0.05)
  • RPE was not significantly different between the three treatments (14.5±0.5 vs. 14.0±0.5 and 14.0±0.5, P≤0.05)
  • Total muscle glycogen re-synthesis over the four-hour recovery period was significantly greater in both CM and CHO treatments than in PLA (23.58mcgmol and 30.58mcgmol per g-1 wet weight vs. 7.05msgmol per g-1 wet weight, P≤0.05); CHO was not significantly different from CM (P=0.06)
  • CM increased the activation status of signaling PROs associated with increased mRNA translation and PRO synthesis compared to PLA. mTOR was greater at recovery minute 45 in CM than in CHO or in PLA (174.4±36.3% vs. 131.3±28.1% and 73.7±7.8% standard, P≤0.05) and at recovery end in CM than in PLA (94.5±9.9% vs. 69.1±3.8%, P≤0.05). rpS6 phosphorylation was greater in CM than in PLA at recovery minute 45 (41.0±8.3% vs. 15.3±2.9%, P≤0.05) and at recovery end (16.8±2.8% vs. 8.4±1.9%, P≤0.05). FOXO3A phosphorylation was greater at recovery minute 45 in CM and in CHO than in PLA (84.7±6.7% and 85.4±4.7% vs. 69.2±5.5%, P≤0.05).
  • No association was demonstrated between improved subsequent performance and reduced muscle damage indicators myoglobin and CPK
  • No treatment differences were detected in any of the protein or anti-inflammatory cytokines
  • There was no statistically significant difference in treatment or in time between the three treatments for ubiquitination
  • Although phosphorylation was significantly greater in both CM and CHO than in PLA (P≤0.05), there was no difference found between CM and CHO. 




Author Conclusion:

CM is more effective than isocaloric CHO or placebo in improving subsequent TT performance in highly trained endurance athletes. CM is also more effective in modulating the activation of key intracellular signaling PROs involved in PRO synthesis during recovery from endurance exercise. 

Funding Source:
National Dairy Council and the National Fluid Milk Processor Promotion Board.
Commodity Group:
Reviewer Comments:
  • Limitations of the study:
    • Small sample size
    • Limited generalizability as the subjects were highly trained cyclists, the type of exercise, recovery time and supplements provided. 
  • Abbreviations:
    • CHO: Carbohydrate
    • PRO: Protein
    • CM: Chocolate milk
    • VO2max: Maximal oxygen consumption
    • TT: Time trial
    • RER: Respiratory exchange ratio
    • PLA: Placebo
    • RPE: Rating of perceived exertion 
    • mTOR: Mammalian target of rapamycin phosphorylation.
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? Yes
  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? Yes
  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? Yes
  3.3. Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.) Yes
  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? Yes
  4.1. Were follow-up methods described and the same for all groups? Yes
  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%.) Yes
  4.3. Were all enrolled subjects/patients (in the original sample) accounted for? Yes
  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.) Yes
  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? Yes
  6.6. Were extra or unplanned treatments described? Yes
  6.7. Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups? Yes
  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? Yes
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)? Yes
  8.5. Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)? Yes
  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