DFA: EPA/DHA and Cognitive Health (2011)

Study Design:
- Click here for explanation of classification scheme.
Quality Rating:
Research Purpose:

To investigate the effect of daily supplementation with high or low doses of EPA and DHA for 26 weeks on cognitive performance in older individuals.

Inclusion Criteria:

Greater than 65 years.

Exclusion Criteria:
  • Score of more than 16 on the Centre for Epidemiologic Studies Depression Scale (CES-D)
  • Score of less than 21 on the Mini-mental State Examination (MMSE)
  • Current or recent (less than four weeks) use of fish oil supplements
  • Intake of more than 400mg of EPA and DHA from fish per day, as estimated from a fish consumption questionnaire
  • Current use of pharmacologic antidepressants or medication for dementia
  • Use of more than four glasses of alcohol per day
  • Additionally, self-reported compliance during the two-week placebo run-in period had to be more than 80%.
Description of Study Protocol:


Mainly recruited through an existing database of volunteers with interest in participating in studies at Wageningen University.  


  • Randomized, placebo-controlled
  • An independent person randomized subjects by means of computer-generated random numbers in stratified permuted blocks of six. Stratification factors included age (less than and 69 or more years), sex, MMSE score (less than and 28 or more) and CES-D screening test score (less than and five or more).

Blinding Used

Double-blind; staff members and participants were blinded toward treatment allocation until completion of blind data analysis.


  • Placebo: High-oleic sunflower oil (contained mainly oleic acid)
  • Fish oil: Daily dose containing either approximately 400mg or approximately 1,800mg EPA and DHA (Lipid Nutrition, Wormerveer, The Netherlands). High dose: 1,093±17mg EPA and 847±23mg DHA. Low dose: 226±3mg EPA and 176±4mg DHA. The mean EPA and DHA ± SD was determined from 20 samples taken at regular times during the study.
  • The oils were administered in soft gelatin capsules each containing 900mg oil and 2.7mg tocopherol as antioxidant. Capsules were packaged in foil strips containing the daily dose of six capsules per strip to facilitate compliance and recording of capsule use (Medipack, The Netherlands).
  • Duration of the intervention was 26 weeks. 

Statistical Analysis

  • Sample size calculation was based on the Word Learning Test, where a difference of four points was considered clinically relevant. With a mean ± SD of 45±8, a minimum sample size of 63 subjects per group would be required to detect a difference (power = 80%, two-sided alpha = 0.05). The investigators included 100 subjects per treatment group, which allowed sub-group analysis and anticipated dropout.
  • Intention to treat was used according to a pre-defined data analysis plan using SPSS
  • A two-sided P-value less than 0.05) was considered significant
  • Baseline characteristics of treatment groups were compared by analysis of variance or Kruskal-Wallis for continuous variables and chi-square for categorical variables
  • Crude cognitive test scores at baseline and after 26 weeks (or 13 weeks) were pooled to calculate the grand mean and SD per test
  • To compare the results of the individual cognitive tests and to limit the number of dependent variables, individual neuropsychological tests were clustered into compound Z-scores for four primary composite endpoints on neuropsychological domains, including sensorimotor speed, memory, executive function and attention
  • To test differential changes among the three intervention groups after 13 and 26 weeks, analysis of variance was used with the treatment group as factor and scores on the different cognitive domains as dependent variables
  • A Dunnett post-hoc test was conducted to compare mean changes in the two treatment groups with changes in the control group
  • After performing the primary analysis, an additional per-protocol analysis was performed, as were pre-planned sub-group analyses for sex and for (non) carriers of the APOE-e4 allele. 
Data Collection Summary:

Timing of Measurements

  • Baseline, 13 and 26 weeks 
  • Subjects were screened between November 2005 and February 2006, intervention took place between February and November 2006.

Dependent Variables

  • Cognitive performance: Five tests with no ceiling effects and are sensitive and robust in detecting small cognitive differences; Word Learning Test measures the storage and retrieval of newly acquired verbal material; forward test of the Wechsler Digit Span Task measures attention and the backward test measures working memory; Trail Making Test version A measures sensorimotor speed and version B measures concept shifting interference (executive function); Stroop Color-Word Test measures selective attention and susceptibility to behavioral interference; Verbal Fluency Test measures the ability to draw on one's encyclopedic memory in a strategy-based manner (executive, verbal reasoning). Participants underwent measurements on the same day of the week and at the same time in the morning, after a standardized small snack in the same quiet room without any obvious distracters.
  • n-3 PUFAs in plasma cholesteryl esters
  • APOE genotype determined by PCR-based restriction fragment length polymorphism method.

Independent Variables

  • Placebo
  • 400mg EPA and DHA
  • 1,800mg EPA and DHA.

Control Variables

  •  Information on medical history, drug use, alcohol consumption, smoking habits, educational level and marital status was obtained by questionnaire
  • Education was categorized according to Statistics Netherlands
  • Physical activity level was estimated by means of a previously described questionnaire
  • Fish intake in the previous three months was estimated with a food frequency questionnaire (60-item list). Items were categorized into three groups based on the amount of fat in the different types of fish, i.e., lean, medium-fat and fatty fish. Information was also obtained on how the fish was consumed. EPA and DHA intake was calculated by multiplying the frequencies of portions of fish per month per group by an EPA and DHA conversion factor (ref 33)
  • Body height, weight and waist circumference.
Description of Actual Data Sample:

Initial N

  • 528 assessed for eligibility
  • 377 willing and eligible for screening visit; 26 excluded for a variety of reasons
  • 351 screened; 26 excluded for MMSE less than 21, CES-D more than 16, or EPA and DHA more than 800mg per day
  • 325 run-in period and  23 withdrew from side effects capsules (two), capsules too big (five); life-threatening disease (one), surgery (two), personal situation (three) and second thoughts (10)
  • 302 randomized  .

Attrition (Final N)

  • Placebo: 106 (56% male)
    • Week 13: Analyzed 105; excluded one due to discontinued intervention
    • Week 26: Analyzed 103; excluded three due to two deceased, one discontinued intervention
  • 400mg EPA and DHA: 100 (55% male)
    • Week 13: Analyzed 100
    • Week 26: Analyzed 100
  • 1,800mg EPA and DHA: 96 (55% male)
    • Week 13: Analyzed 95; excluded one discontinued intervention
    • Week 26: Analyzed 96; excluded none. 


  • Placebo: 70.1±3.7
  • 400mg: 69.5±3.2
  • 1,800mg: 69.9±3.4

Other Relevant Demographics

  • Married or living together (percentage): Placebo, 77; 400mg, 81; 1,800mg, 80
  • Education, low/middle/high (percentage): Placebo, 5/59/37; 400mg, 11/49/40; 1,800mg, 10/54/35, respectively 
  • Smokers (percentage): Placebo, 10; 400mg, eight; 1,800mg, eight
  • Ex-smokers (percentage): Placebo, 56; 400mg, 54; 1,800mg, 64
  • Never smokers (percentage): Placebo, 34; 400mg, 38; 1,800mg, 28
  • Alcohol consumers (percentage): Placebo, 88; 400mg, 87; 1,800mg, 80
  • Median alcohol consumption (glasses per week): Placebo, 10 (six to 14); 400mg, eight (four to 14); 1,800mg, eight (four to 14)
  • Coffee consumers (percentage): Placebo, 97; 400mg, 96; 1,800mg, 96.


  • BMI (kg/m2): Placebo, 26.5±3.9; 400mg, 26.2±3.4; 1,800mg, 26.1±3.0
  • Waist circumference (cm): Placebo, 95.9±12.1; 400mg, 94.2±10.6; 1,800mg, 94.5±11.4
  • Physical activity score: Placebo, 11.4±6.2; 400mg, 11.1±6.2; 1,800mg, 11.5±6.4 


Netherlands; research center at Wageningen University; subjects free-living.

Summary of Results:

Key Findings

  • Baseline:
    • Fish consumption (times per month): Placebo, six (four to eight); 400mg, five (three to nine); 1,800mg, seven (four to nine)
    • EPA and DHA intake (mg per day): Placebo, 316 (166 to 584); 400mg, 278 (103 to 487); 1,800mg, 306 (131 to 592)
    • Plasma EPA and DHA (mass percentage): Placebo, 1.9±1.1; 400mg, 1.9±1.1; 1,800mg, 1.9±0.9
    • APOE-e4 allele (0/1/2 percentage, respectively): Placebo, 71/26/2; 400mg, 69/30/1; 1,800mg, 68/29/3
    • MMSE score: Placebo, 28 (27 to 29); 400mg, 28 (27 to 29); 1,800mg, 29 (27 to 29)
    • MMSE range: Placebo, 23 to 30; 400mg, 24 to 30; 1,800mg, 23 to 30
    • Self-perceived memory impairment (percentage): Placebo, 68; 400mg, 50; 1,800mg, 53 (significant difference among groups)
  • Intervention:
    • In general, cognitive test scores in all three groups improved, but changes were not significantly different among the groups and were probably mostly due to learning effects because scores in the placebo group increased to the same extent (data is shown in Table 2 of the article)
    • After 13 and 26 weeks of supplementation, there were no significant differential changes for fish oil vs. placebo in any of the cognitive function domains except for a transitory change in memory
    • An interaction effect between treatment and APOE genotype on the cognitive domain of attention was observed. APOE-e4 carriers in the 400mg group (N=31; P=0.03) and in the 1,800mg group (N=31; P=0.04) showed an improvement after 26 weeks of intervention compared with placebo
    • An interaction effect was also demonstrated between treatment and sex on the cognitive domain of attention, i.e., an improvement in men (N=167; P=0.05) after 26 weeks of supplementation with 400mg compared with the placebo.

Other Findings

  • Adherence to treatments based on counts of returned capsules was high (99%, with only three subjects less than 80%) and did not differ among treatment groups
  • Compliance was confirmed by a change in the proportion of EPA and DHA in plasma cholesteryl esters of +51% in the low-dose and +236% in the high-dose group. The placebo had a change of -2%
  • Main complaints concerned mild gastrointestinal discomfort
  • At the end of the study, blinding of subjects toward treatment allocation (fish oil, placebo or no idea) was evaluated. The proportion of participants who thought they had received fish oil or placebo did not differ among the groups. 
Author Conclusion:
  • A limitation as cited by the authors: Duration of the intervention may have been too short
  • No effect of EPA and DHA supplementation for 26 weeks on cognitive performance in healthy older individuals was observed
  • The data suggests that in subjects carrying the APOE-e4 allele and in male subjects, EPA and DHA may improve attention
  • Based on these findings, longer-term EPA and DHA supplementation studies to investigate effects on cognitive performance are warranted, especially in groups at higher risk for cognitive decline.
Funding Source:
Government: The Netherlands Organization for Health Research and Development
Reviewer Comments:
  • In the text of the Study Design section, the authors state they were able to include 100 subjects per treatment group. However, as shown in Figure one, there were not 100 subjects per group. This difference could be due to the stratification used during the randomization process.
  • Figure one showing flow of participants through the study: In the placebo group at both weeks 13 and 26, participants were excluded from analysis because of discontinued intervention. However, for the 1,800mg EPA and DHA treatment group, at week 13 one participant was excluded from analysis because of discontinued intervention but at week 26 that participant appears to have been added back, as the N goes back up to 96. An intent-to-treat analysis was supposed to have been used. This should have also applied to the placebo group, so it is unclear if there is a mistake in figure one or if the information is correct regarding the N-per-group for analysis.
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? Yes
  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? Yes
  6.3. Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect? ???
  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? ???
  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? ???
  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? Yes
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