DFA: Conjugated Linoleic Acid (CLA) Supplementation and Intermediate Health Outcomes (2011)

Study Design:
- Click here for explanation of classification scheme.
Quality Rating:
Research Purpose:
  • To evaluate the efficacy of creatine monohydrate (CrM) supplementation as an adjunct to six months of moderate intensity resistance exercise training on measures of body composition, functional tasks and strength in a cohort of older adult men and women
  • To determine whether CLA leads to a reduction in body fat during resistance training and to evaluate safety issues related to oxidative stress and hepatic dysfunction in older adults
  • To evaluate whether resistance exercise training with or without a CrM+CLA supplementation would create favorable alter markers of inflammation, oxidative stress, blood lipids or adipokines in older adults.
Inclusion Criteria:
  • Community dwelling, older adults (more than 65 years of age)
  • Did not participate in sports training programs or performed vigorous physical activity more than three times per week, more than 30 minutes per day, in the year prior to the commencement of the study
  • Did not participate in resistance exercise activities in the preceding two years
  • Did not require the use of assistive devices for mobility
  • Women were post-menopausal.
Exclusion Criteria:
  • Evidence of coronary heart disease
  • Congestive heart disease
  • Uncontrolled hypertension
  • Chronic obstructive pulmonary disease
  • Diabetes mellitus
  • Renal failure
  • Major orthopedic disability
  • Smoking
  • Hormone replacement therapy
  • After the initial screening, the subjects were required to perform a cycle ergometry test to five METS (metabolic equivalents) on a mechanically braked cycle ergometer. A 12-lead electrocardiogram was taken prior to and upon completion of the ergometry test, as part of exclusion criteria to participate in the resistance exercise training program. Any participants with abnormal resting ECG or a blood pressure of higher than 160mm Hg systolic or 95mm Hg diastolic were not allowed to complete the screening tests.
Description of Study Protocol:


  • Recruited from within a 30km radius of the research center
  • Recruitment strategies included local newspaper ads, radio announcements and flyers distributed to surrounding areas
  • Each subject underwent a thorough screening, which included a telephone interview and a medical evaluation.


  • Randomized, double-blind, placebo-controlled
  • Randomization was done by having recruited men and women randomly draw from two blocks (one male and one female) each containing 24 shuffled and sealed envelopes with each block having equal allocation to treatment (N=12 per block) or placebo (N=12 per block).

Blinding Used

  • Double-blinded
  • The flavor and appearance of the supplements were indistinguishable by the subjects and investigators.


  • Placebo (PL): 7g dextrose per day + 6g of safflower oil per day
  • CrM+CLA: 5g CrM (Neotine) + 2g dextrose per day (Palo Alta, CA); and 6g CLA (CLA-ONE 45% c9, t11; 45% t10, c12, Pharmanutrients, Gurnee, IL)
  • Intervention period was for 24 weeks.

Statistical Analysis

  • Subject characteristics were analyzed using an unpaired T-test (age and height)
  • In accordance with NIH guidelines, and given that sex differences in sarcopenia have been reported, the statistical analysis was planned to determine whether or not sex differences existed
  • For the main outcome variables of body composition (FFM and body fat) and strength (knee extension and handgrip), a Kolmogorov-Smirnov test was first run and the distribution plots reviewed to determine whether the data was normally distributed. In all cases the criteria for a normal distribution were met and the outcome variables analyzed using a three-way analysis of variance with sex a between variable (men vs. women), and training (pre- vs. post-) and supplement (CrM+CLA vs. placebo) as the repeated measures variable. 
  • A Tukey's HSD post-hoc analysis for unequal sample sizes was used to locate pair-wise differences when statistical significance was observed
  • In addition, body composition measures were further analyzed to blood and urine markers using Pearson R correlation
  • Significance was set at P
  • Values are reported as mean ± standard deviation (SD).
Data Collection Summary:

Timing of Measurements

  • Resistance training was twice per week and all participants recorded in a log book each session
  • Once recruited, each subject was put through three separate visits for pre- and post-testing
  • Diet records were collected one week prior to the start and during the final week of exercise training
  • Urine samples were collected before training began and once again in the last week of training
  • Blood samples (overnight post-absorptive) were collected between 8:00 and 9:30 A.M. before training began and once again in the last week of training. 

Dependent Variables

  • Functional testing:
    • Five functional tasks were performed once using a stopwatch
    • 30-meter walking: Subjects walked as fast and safely as possible for a 30m distance that was marked off with clear start and stop points
    • Balance: Subjects were timed during a 'heel to toe' tandem gait on a pre-marked 9.14m tape line on the floor. Subjects could keep their arms out to aid in balance and for safety. Any step off the line resulted in a one second addition to the total task time.
    • Sit to stand: Subjects performed a series of consecutive rising and sitting from a sturdy, armless plastic chair secured against a wall. Starting from a seated position, the numbers of completed repetitions were recorded over 30 seconds, with their arms at their sides or across their chests while performing the task.
    • Chair rise and walk: Starting from a seated position, subjects stood up and walked as quickly as possible in a pre-determined straight line to a pylon at 9.14m, go around the pylon and return to their original seated position
    • Stair climb: Subjects climbed two flights of stairs (N=14), starting with both feet on the bottom platform and ascending one step at a time, using a handrail only if insecure
  • Strength testing:
    • Isometric ankle dorsiflexion: Maximal ankle dorsiflexion was taken as the highest of three trials of five seconds maximal contractions using a custom-made isometric device with the ankle joint at 90o
    • Hand grip strength: Measured using a isometric dynamometer. Subjects performed three x five seconds with a one-minute rest between each trial
    • Isometric and isokinetic knee extension: Measured using a dynamometer. Subjects were positioned into the machine with the knee flexed at 90o and performed three x five-second maximal voluntary contractions with 30 seconds between each trial at each speed
    • IRM and endurance testing: One repetition maximum (one RM) testing occurred prior to training commencement and post-training for use as an outcome measurement and to set individual weight assignments for the training sessions, including leg press, chest press, arm flexion and leg extension. Muscle endurance was also completed at the end of the study using the weight from the initial one RM by determining the number of repetitions done post-training using the original one RM weight.
  • Body composition:
    • Body mass and height were measured to the nearest 0.1kg and 0.5cm, respectively, using a calibrated electronic scale
    • Body composition was assessed using dual energy x-ray absorptiometry scan (Hologic). Fat free mass (FFM), fat mass (FM) and bone mineral density (BMD) of the entire body, spine and femur were recorded.
  • Blood and urine analyses:
    • Serum glucose, creatine kinase (CK), bilirubin, gamma glutamyl transferase (GGT), LDL, HDL and total cholesterol were each analyzed in batches containing samples from men and women, pre- and post-training and from each of the treatment groups
    • All other metabolites were analyzed using ELISA-based assays for leptin, C-reactive protein (CRP), adiponectin, and interleukin-6 (IL-6)
    • Insulin concentrations were measured by radioimmunoassay. The HOMA-IR index was calculated using the equation [fasting serum insulin (uU/ml) x fasting plasma glucose (mmol per L)] divided by 22.5
    • Urine creatine (Cr) and creatinine (Crn) were analyzed using high-performance liquid chromatography. The creatinine clearance rate was calculated from the measured serum and urinary creatinine values using the equation [urine creatinine (mg per dL) x urine volume (ml per 24 hours)] divided by [1,440 x serum creatinine (mg per dL)]
    • Urinary 8-isoprostanes (8-isoP), 8-hydroxy-2-deoxy-guanosine (8-OH-2dG) and N-telopeptides were analyzed by ELISA.

Independent Variables

  • Sex (between variable): Men vs. women
  • Training [repeated measures (pre- vs. post-)]: Resistance exercise training program twice per week for six months, while being supervised in a group environment for each training session. All participants recorded each contraction in a log book. Each training session was preceded by a five-minute aerobic warm-up of spinning on a cycle ergometer or brisk walking, followed by a brief stretching session. 12 exercises were used to train the major muscle groups of the upper and lower body in a circuit-set system using weight training machines. Subjects performed 12 repetitions of each exercise, including leg press, chest press, leg extension, should press, lateral pull-down seated row, calf raise, abdominal crunch, back extension and 10 repetitions for arm flexion and arm extension. The training protocol progressed from performing one set of each exercise at 50% of their one-RM values up to three sets of 75% of their one-RM values over the course of the training period. The one-RMs were retested every four weeks to accommodate any increases in strength by the subjects and readjusted to the new one-RM value.
  • Supplement (repeated measures): Placebo (7g dextrose per day + 6g of safflower oil per day); CrM+CLA (5g CrM (Neotine) + 2g dextrose per day) and 6g of CLA (CLA-One 45% c9, t11; 45% t 10, c12, Pharmanutrients).
Description of Actual Data Sample:
  • Initial N:
    • See Figure 1 of article for a summary diagram of the recruitment strategy and allocation
    • 76 subjects were assessed for eligibility (N=76)
    • After six months of recruitment and a lack of drop-outs, recruitment was closed with a total of 19 men and 20 women
  • Attrition (final N): 19 men, 20 women; none were lost to follow-up or discontinued intervention. None were excluded from analysis.
  • Age:
    • Placebo: Men, 74.8±6.6; women, 68.3±4.4
    • Supplement: Men, 71.8±5.2; women, 69.5±3.8
  • Other relevant demographics: Lived within a 30km radius of the research center (Canada)
  • Anthropometrics (pre-training):
    • FFM (kg):
      • Placebo: Men, 55.8±5.5; women, 41.1±5.1
      • Supplement: Men, 58.1±8.6; women, 41.3±4.4
    • FM (kg):
      • Placebo: Men, 18.8±4.4; women, 22.8±5.7
      • Supplement: Men, 21.4±4.9; women, 23.5±5.3
    • BMI:
      • Placebo: Men, 25.9±2.7; women, 25.2±3.0
      • Supplement: Men, 27.8±2.1; women, 25.5±3.1
    • BMD-total:
      • Placebo: Men, 1.21±0.09; women, 1.13±0.12
      • Supplement: Men, 1.20±0.13; women, 1.02±0.09
    • BMD-hip:
      • Placebo: Men, 1.01±0.09; women, 0.86±0.14
      • Supplement: Men, 0.94±0.10; women, 0.83±0.10
    • BMD L-spine:
      • Placebo: Men, 1.12±0.06; women, 1.12±0.15
      • Supplement: Men, 1.19±0.14; women, 0.94±0.11
  • Location: Within 30km radius of research centre at McMaster University, Hamilton, Ontario, Canada.
Summary of Results:

 Key Findings

  • Resistance exercise training increased FFM more for men (+2.0kg, P<0.001) compared to women (+1.0kg, P<0.05), with a significantly greater increase in FFM for the CrM+CLA group (+2.1kg, P=0.02) compared to placebo (+0.9kg, P+0.06, NS)
  • Significantly greater reduction in fat mass in the CrM+CLA group following training (-1.9kg, P<0.001) as compared to placebo (-0.4kg, NS)
  • Lumbar BMD was lower for women but did not change in response to exercise training with or without CrM+CLA
  • Every measure of muscle strength (P<0.05 to P<0.001) increased following exercise training
  • The CrM+CLA supplement did not further enhance isometric strength measurements after training; however, isokinetic strength increased more after training for those on supplement vs. placebo (P<0.05)
  • Women on CrM+CLA showed a greater increase in knee extension one-RM strength after training as compared to all other groups (P<0.05)
  • Muscle endurance increased significantly (P<0.0001) following training with the improvements in chest press and arm flexion showing a greater improvement (P<0.05), with a trend towards a greater improvement in leg press (P=0.077), for the CrM+CLA group
  • There was a three-way interaction (P<0.05) with women showing a greater improvement in knee extension endurance after training for the CrM+CLA group
  • Exercise training improved all five measures of functional capacity for men and women with no differential effect of CrM+CLA
  • Following resistance exercise training, there was an increase in CK activity, and an increase in total and LDL-cholesterol (P<0.05), without a change in LDL:HDL ratio
  • As expected, an increase in serum creatinine was seen following training only in the CrM+CLA group (P<0.05)
  • Urinary creatine, creatinine and Cr/Crn ratios were all higher following training only in the Cr+CLA supplemented group. Neither resistance training, nor the CrM+CLA supplement, influenced the measured plasma creatinine clearance. The 8-OH2-dG/creatinine ration was lower for both groups following training.
  • The 8-isoP:creatinine ratio was higher following training only for the females in the CrM+CLA group
  • There were no effects of sex, exercise training or CrM+CLA supplementation on urinary N-telopeptide excretion
  • Subjects tolerated the supplementation protocol well, with only one report of gastrointestinal distress (stomach cramps) on CrM+CLA, but this did not lead to discontinuance of the study
  • There were no reports of muscular cramping or any other subjective symptoms during the study.

Selected Results on Body Composition

Men Placebo Supplement
  Pre-test Post-test Pre-test Post-test
FFM (kg




FM (kg)  18.8±4.4  18.2±4.2  21.4±4.9  19.9±5.9** 
Women Placebo   Supplement  
  Pre-test  Post-test  Pre-test  Post-test 
FFM (kg) 41.1±5.1  41.5±5.1  41.3±4.4  42.9±4.7** 
FM (kg)  22.8±5.7  22.6±5.9  23.5±5.3  21.5±5.6** 

*Indicates a significant interaction between men and women in response to training.

**Indicates a significant interaction between training and supplement.

Other Findings

  • The men were slightly older, taller, heavier and had higher FFM compared to the women
  • Men consumed more energy compared to women (P<0.02), but there was no effect of training on energy intake, and men on placebo had lower percentage fat intake vs. all other groups. There were no other sex, training or supplement effects on dietary composition.
  • Total bone mineral density (BMD) was lower for women and did not change after training. Hip BMD was lower for women and decreased slightly, but significantly for men only after training (interaction, P<0.05).
  • All measures of strength training were higher for men compared to women
  • Correlation analysis showed that those with the lowest initial knee extension strength had the greatest percentage increase in strength after training (R=-0.61, R2=0.37, P<0.05)
  • Women had slightly worse balance walk scores and sit to stand performance compared to men
  • Women had higher leptin, adiponectin and HDL-C concentrations than did men (P<0.001) and lower creatinine and bilirubin concentrations as compared with men
  • Women had higher 8-OH-2dG:creatinine and Cr:Crn ratios and lower 24-hour creatinine compared to men
  • No individual dropped out due to musculoskeletal injuries.
Author Conclusion:
  • Confirmed the beneficial effects of resistance training on increasing FFM, strength and function in older adults and have added more data showing that a combination of CrM+CLA lead to greater increases in FFM and losses of fat mass
  • Furthermore, those on the supplement showed greater muscular endurance and no evidence of clinical or serological side effects
  • Whether such changes are maintained in the longer term is unclear at this point.
Funding Source:
Government: Canadian Institute of Health Research
Reviewer Comments:
  • The author's mention three separate visits for pre- and post-testing. It is unclear what exactly the three visits were but based on the provided information, it appears these were:
    • The cycle ergometry test performed after the initial screening
    • A time point prior to the beginning of training for collection of blood and urine samples
    • Post-test during the last week of training
  • It was stated there was no attrition and no subject was excluded from analysis. This means for every outcome variable the total N should be 39 (18 placebo and 21 supplement, as shown in Figure 1). However, Table 2 Dietary Analysis only shows results from 12 placebo and 17 supplement and Table 3 muscle strength tests only shows 20 supplements. It is unclear why these numbers are different.
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? 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? 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)? No
  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