NAP: Recovery (2014)


Rowlands DS, Wadsworth DP. Effect of high-protein feeding on performance and nitrogen balance in female cyclists. Med Sci Sports Exerc. 2011; 43 (1): 44-53.

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 effect of protein quantity ingested during recovery from high-intensity exercise on recovery and subsequent performance and to estimate dietary protein requirement during intensive training in well-trained women.

Inclusion Criteria:

Female endurance trained cyclist.

Exclusion Criteria:

Precluding medical conditions.

Description of Study Protocol:


Not described.


  • Randomized, crossover trial
  • On Day One, subjects consumed standard low-PRO animal flesh-free diet, with the last meal four hours pre-exercise. In the lab, 2.5-hour cycling intervals were performed (average work, 7.5mJ)
    • 12-minute warm-up at 30% Wmax, five minutes at 40% Wmax, five minutes at 50% Wmax
    • 10 two-minute intervals at 90% Wmax
    • 12 two-minute intervals at 80% Wmax
    • Intervals alternated with two-minute recovery periods at 50% Wmax and finished with three five-minute intervals at 70% interspersed with three five-minute intervals at 50% Wmax  
    • During all exericise procedures, subjects were provided with 6.8% CHO-electrolyte solution to provide CHO at the rate of 0.8g per kg fat-free mass (FFM) per hour.

Blinding Used 



  • Protein-rich recovery feeding intervention (high-protein) and an isocaloric low-protein control condition (control). Bar and dinrk servings were given every 30 minutes, starting immediately after exercise on Day One for four hours, totaling eight bars and drinks.  
  • High-protein was a chocolate-coated bar and milk-like drink formulation (chocolate or vanilla-flavored). High-protein bar provided 1.6g CHO, 0.8g protein and 0.29g fat per kg FFM per hour. 
  • Low-protein was an isocaloric bar and milk-like drink formulated to resemble high-protein. Control bar provided 2.35g CHO, 0.12g protein and 0.29g fat per kg FFM per hour. 

Statistical Analysis

  • The effect of protein dose on dependent outcomes was estimated from mixed modeling
  • Most dependent outcomes were analyzed after log transformation to accommodate the heteroscedastic distribution of residuals and to express differences as percentages
  • Outcomes were determined from the interaction between the following fixed effects: Treatment, order of treatment (treatment 1 or 2), day (Day Two or Four)
  • For the analysis of sprint performance, lactate and perpetual responses, sprint number or time was coded as a quantitative number predictor (as in linear regression) to model the effect of time on outcomes (slope and fatigue effects)
  • For the analysis of creatine kinase and nitrogen balance, estimates were derived from repeated-measures models (as in traditional ANOVA). 
Data Collection Summary:

Timing of Measurements

  • Blood sample five minutes after exercise and every 30 minutes for the following 120-minute recovery with a final sample at 180 minutes on Days One and Four
  • On Day Two, blood at baseline, 15 minutes after small breakfast and every 30 minutes for the first 90 minutes into recovery
  • Urine collection from completion of exercise on Day One until the beginning of exercise on Day Four.

Dependent Variables

  • Ratings of fatigue and exertion: Visual analog scales immediately after priming intervals two and five and sprints one, four, seven and 10. Measured categories were sensations of tiredness, leg soreness, ability to sprint, level of exertion and nausea.
  • Plasma lactate and glucose: Automated analyzer
  • Creatine kinase: Enzymatic assay
  • Urinary urea: Spectrophotometry
  • Urinary creatine and creatinine: High-performance liquid chromatography
  • Nitrogen balance: Calculated during three periods to total 60 hours:
    1. From completion of cycling on Day One to final urine collection shortly before the pre-exercise meal on Day One
    2. During exercise on Day Two
    3. From the end of exercise on Day Two to the final urine collection on morning of Day Four preceding the pre-exercise meal.
  • Nitrogen losses were from sweat at rest, sweat during exercise on Day Two and from feces and miscellaneous loss throughout the 60-hour collection.   

Independent Variables

  • Protein-rich recovery feeding intervention (high-protein) and an isocaloric low-protein control condition (control)
  • High-protein was a chocolate-coated bar and milk-like drink formulation (chocolate or vanilla-flavored).

Control Variables

Two four-day experimental blocks separated by 28 days to control for effect of menstrual cycle, with the first block beginning three to seven days after the first day of menstruation

  • On Day One: Standardized animal flesh-free diet four hours before testing. Fatigue-inducing ride of 2.5-hour intervals with an average estimated total work performed of 7.5mJ. Recovery food was consumed in units immediately after the first blood draw and in 30-minute intervals for the first three hours, with the last unit consumed one hour after leaving the lab.
  • Day Two: Standardized ride followed by a sprint performance (3.6mJ). Ingestion of recovery food same as on Day One. Standardized CHO-rich diet that was provided was then started and followed until late evening of Day Three.
  • Day Four was identical to Day Two.
Description of Actual Data Sample:
  • Initial N: 12 females
  • Attrition (final N): 12
  • Age: 30±7 years
  • Ethnicity: Not described
  • Other relevant demographics
    • V02max: 3.4±0.4L per minute
    • Peak power output: 260±26W
    • Training for 4.9±4.1 years with a weekly average training of 12.8±3.8 hours during the previous six months.
  • Anthropometrics
    • ?FFM: 49.5±3.1kg
    • Body fat mass: 19±3%
    • Body mass: 60.8±3.4kg.
  • Location: New Zealand.
Summary of Results:


  • No clear effect of protein dose on overall sprint mean power. The differences in decline in mean power from sprint one to 10 were also inconclusive of both days.
  • With the treatment effect factored out, overall sprint mean power on Day Four compared with Day Two was 4.2% higher (95% CI, ±3.2%) during the first and 4.9% (±3.3%) during the second experimental block, respectively. The second order of trial increased overall sprint mean power on Days Two and Four relative to the first by an insubstantial 0.7% (±4.6%) and 1.4% (±4.6%), respectively.   
  • A possible substantial increase in perceived effort of 0.4U (±0.8U) was observed in high-protein during pre-load priming intervals on Day Two. During sprints on Day Two, cyclists demonstrated likely and very likely elevations in perception of tiredness and leg soreness, respectively, coupled with an almost-certain decrease in perceived leg strength in high-protein relative to control. During the pre-load priming intervals in Day Four, feelings of tiredness (0.5±0.8U) and soreness (0.5±0.7U) were possibly elevated and effort likely substantially elevated (0.6±0.8U) in high-protein; conversely, a likely substantial decrease in nausea (-0.3±0.3U). During sprints on Day Four, likely and possible increases in leg soreness and tiredness were observed in high-protein, respectively. 
  • Moderate (14±5%) and large (22±10%) increase in glucose in control during recovery on Days One and Two, respectively. Effects on glucose during sprints were trivial, being 4% lower (±4%) and 1% higher (±6%) with high-protein and Days Two and Four, respectively. 
  • Plasma lactate was 24% lower (±12%) during recovery on Day Two in high-protein. During sprints, there were moderate (28±18%) and small (20±22%) reduction in lactate in high-protein on Days Two and Four, respectively. 
  • Effects of diet on plasma creatine kinase activity were unclear or trivial throughout
  • Nitrogen balance was positive in high-protein but negative in control; the result in difference between conditions was 247mg of N per kg FFM (95% CI, ±47mg of N per kg FFM). During exercise on Day Two, cyclists were in neutral balance in control but slightly negative in high-protein and from Day Two to the morning of Day Four, cyclists were in slightly positive nitrogen balance in high-protein and slightly negative in control, with a net 34mg of N per kg FFM more positive balance (±48mg of N per kg FFM) in high-protein. The nitrogen balance regression produced an estimate average dietary protein requirement of 1.28g per kg per day (95% CI, 0.54g to 2.67g per kg per day) to achieve zero nitrogen balance.
Author Conclusion:
  • No clear influence of dietary protein quantity on the subsequent performance in females
  • The findings on nitrogen balance suggest that female cyclists training intensely have daily protein requirements approximately 1.6 times the recommended daily allowance but 0.65 times that of males.
Funding Source:
University/Hospital: Massey University, Wellington, Australia
Reviewer Comments:
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? Yes
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? 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.) 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? 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? 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)? 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