Vitamin D and Omega-3 Trial (VITAL)
Phase: PHASE3
Conditions: Cancer, Cardiovascular Disease
Sponsor: Brigham and Women's Hospital

PROTOCOL 
 
 
Official title of study:  VITamin D and OmegA-3 TriaL (VITAL) 
NCT #: NCT01169259 
Date: November 30, 2017 
 

Protocol
This trial protocol has been provided by the authors to give readers additional information about their work.
Protocol for: Manson JE, Cook NR, Lee I-M, et al. Vitamin D supplements and prevention of cancer and cardio-
vascular disease. N Engl J Med 2019;380:33-44. DOI: 10.1056/NEJMoa1809944

Vitamin D Supplementation and Primary Prevention of Cancer and Cardiovascular 
Disease: Principal Results from the VITamin D and OmegA-3 TriaL 
 
 
 
 
This supplement contains the following items: 
 
1. Original protocol, final protocol, summary of changes. 
 
2. Original statistical analysis plan, final statistical analysis plan, summary of 
changes. 

 
1
 
 PROTOCOL FOR THE VITAL TRIAL  
January 28, 2010 
 
 
 
SUMMARY 
 
The VITAL trial (VITamin D and OmegA-3 TriaL) is a randomized, double-blind, placebo-
controlled, 2x2 factorial trial of vitamin D (in the form of vitamin D3 [cholecalciferol]) and marine 
omega-3 fatty acid (eicosapentaenoic acid [EPA] + docosahexaenoic acid [DHA]) supplements 
in the primary prevention of cancer and cardiovascular disease (CVD). The trial will be 
conducted among 20,000 men aged ≥60 and women aged ≥65, to be recruited from  mailings to 
2.5 million persons, including health and other professionals, members of AARP (formerly 
known as the American Association of Retired Persons), and others. Willing and eligible 
respondents to the mailings will be enrolled in a 3-month run-in, during which they will receive 
placebos. Upon successful completion of the run-in, participants who remain willing and eligible 
will be randomly assigned to one of four treatment groups for 5 years: vitamin D3 (2000 IU/d) 
and fish oil (1000 mg capsule containing 840 mg EPA+DHA); vitamin D3 and fish oil placebo; 
placebo vitamin D3 and fish oil; and placebo vitamin D3 and placebo fish oil. Blood samples will 
be collected and stored to allow assessment of effect modification by baseline 25-
hydroxyvitamin D and omega-3 fatty acid levels, as well as future ancillary studies of 
genetic/biochemical hypotheses. At 1-year intervals, participants will receive a new supply of 
pills and a follow-up questionnaire on compliance, possible side effects, and incidence of 
endpoints. Endpoints will be confirmed by medical record review.  
I. BACKGROUND AND SIGNIFICANCE 
The VITAL Trial will evaluate the role of vitamin D and long-chain marine omega-3 
polyunsaturated fatty acid (eicosapentaenoic acid [EPA] plus docosahexaenoic acid [DHA]) 
supplements in the primary prevention of cancer and cardiovascular disease (CVD). Although 
there have been marked advances in our understanding of potentially beneficial effects of 
vitamin D and omega-3 fatty acids on cancer and cardiovascular risk in recent years, clear gaps 
in knowledge remain.  
Data from laboratory studies, epidemiologic investigations, and small clinical trials strongly 
suggest a protective effect for vitamin D against cancer, but there are no completed or ongoing 
large randomized trials of high-dose vitamin D supplements for the primary prevention of cancer 
in a general population.1 At a 2007 conference on the role of vitamin D in cancer prevention 
sponsored by the National Cancer Institute (NCI) and the National Institutes of Health Office of 
Dietary Supplements (ODS), most speakers “expressed a need for intervention studies with 
comprehensive assessment of vitamin D status.1 To quote one such investigator, Dr. Edward 
Giovannucci, “Further observational studies—and randomized trials if feasible—would be useful 
in testing the hypothesis that vitamin D lowers cancer risk. … Confirming that vitamin D reduces 
the risk of cancer incidence or mortality is critical, because current health recommendations 
typically do not encourage high intakes of vitamin D and tend to discourage sun exposure. 
Current dietary recommendations are geared only to prevent quite low vitamin D levels. If the 
association between better vitamin D status and reduced cancer risk is a causal one, the levels 
of intake currently recommended are probably inadequate. [Note: Institute of Medicine 
guidelines call for 400 IU/d of vitamin D for adults aged 51-70 and 600 IU/d for adults aged >70 
to maintain bone health and normal calcium metabolism.2] Defining … the optimal levels of 

 
2
vitamin D for cancer prevention remains a challenge, but further study should be a high priority 
because the potential for benefit is substantial.”3 
Available data on the connection between vitamin D and CVD are less extensive but also 
support a possible protective effect. It is thus concerning that >1/2 of U.S. middle-aged and 
older women and >1/3 of similarly aged men have vitamin D insufficiency.4, 5  African-American 
(black) individuals are particularly vulnerable, because darkly pigmented skin is less able to 
synthesize vitamin D in response to solar radiation and because they tend to have lower intakes 
of dietary and supplemental vitamin D than whites.6, 7 Obese individuals are also at above-
average risk, presumably due to decreased bioavailability of this fat-soluble vitamin.8, 9 Given 
the aging population and soaring obesity prevalence,10, 11 low vitamin D status is an increasingly 
important public health issue. 
Marine omega-3 fatty acids have shown considerable promise for the primary prevention of 
CVD in laboratory and observational studies; large randomized trials in secondary prevention or 
high-risk settings also find benefit. However, there are no completed or ongoing trials of such 
supplements for the primary prevention of CVD in a general population that has been selected 
only on the basis of age and not on vascular risk factors such as diabetes. The Nutrition 
Committee of the American Heart Association (AHA) has stated that “Additional studies are 
needed to confirm and further define the health benefits of omega-3 fatty acid supplements for 
… primary … prevention.”12 (p. 2775) The conclusion from a 2004 workshop of the ODS and the 
National Heart, Lung and Blood Institute (NHLBI) Working Group on Future Clinical Research 
Directions on Omega-3 Fatty Acids and CVD13 is that “… the body of evidence is consistent with 
the hypothesis that intake of omega-3 fatty acids reduces CVD but … a definitive trial is needed” 
and that “A primary prevention trial [would] fill a major gap in knowledge because virtually none 
of the trials to date have been conducted in a primary prevention population and thus … it is 
unknown whether omega-3 fatty acids can prevent first events.” In terms of methodology, the 
ODS/NHLBI Working Group recommended a large simple trial, with fish oil given in supplement 
rather than in food form. It is critically important that such a trial be undertaken in a timely 
fashion, to avoid a possible dilution of effect from fortification of the food supply with omega-3 
fats, an increasingly common practice among U.S. manufacturers.14  
It is important to clarify the roles of vitamin D and omega-3 fatty acids in the primary prevention 
of cancer and CVD because their purported health benefits are receiving increasing attention in 
both the medical literature and the popular press.15-31 The Canadian Cancer Society recently 
raised its recommended dose of vitamin D to 1000 IU/d,32 and many experts are urging a similar 
move in the U.S.33 Sales of individual vitamin D supplements at U.S. grocery, drug, and “big 
box/club” stores jumped 91% to nearly $30 million during 2007 (C. Reider of Pharmavite LLC, 
personal communication). In 2007, sales of omega-3 fatty acid supplements in the U.S. reached 
$600 million, an increase of 20% from the prior year; omega-3 supplements are now the fifth-
best-selling dietary supplement.14 In addition, an increasing number of foods are omega-3 
fortified; >1200 such products were launched in 2006 alone.14 The growing enthusiasm for 
vitamin D and fish oil underscores the urgent need for a timely initiation of a large randomized 
trial to test these agents rigorously, before their use becomes so prevalent (through 
supplements and fortification of food) as to render participant recruitment and hypothesis testing 
impossible. As Dr. Cindy Davis of the NCI has noted, “While vitamin D may well have multiple 
benefits beyond bone, health professionals and the public should not in a rush to judgment 
assume that vitamin D is a magic bullet and consume high amounts of vitamin D. More definitive 
data on both benefits and potential adverse effects of high doses are urgently needed.”34 The 
same can be said of marine omega-3 fatty acids; the conclusion from a 2004 workshop on 

 
3
omega-3 fatty acids and CVD sponsored by ODS and the NHLBI was that “a definitive trial is 
needed.”13  
Indeed, skeptics will note that most single-agent nutritional interventions have not withstood the 
test of rigorous trials for the primary prevention of cancer and CVD—witness the disappointing 
findings for antioxidant vitamins.35-40 However, unlike the antioxidant scenario, the two agents in 
this proposal appear promising not only in observational studies but also in randomized settings. 
Omega-3 fatty acids have shown cardiovascular benefit in two large trials in high-risk 
individuals41, 42 (one of which also tested vitamin E and found no benefit for it42), while vitamin D 
was suggestive of cancer benefit in a modestly sized trial43 and of reductions in total and cancer 
mortality even at a low dose in another.44 The proposed large-scale randomized controlled trial 
of vitamin D and omega-3 fatty acids will provide definitive data on their roles in the primary 
prevention of cancer and CVD. 
II. SPECIFIC AIMS 
 
The overall aim of the VITAL trial is to evaluate the role of vitamin D and long-chain marine 
omega-3 polyunsaturated fatty acid (eicosapentaenoic acid [EPA] plus docosahexaenoic acid 
[DHA]) supplements in the primary prevention of cancer and cardiovascular disease (CVD). 
Primary aims: 
1. To test whether vitamin D3 supplementation reduces the risk of (a) total cancer 
(excluding non-melanoma skin cancer) and (b) major CVD events (a composite endpoint 
of myocardial infarction [MI], stroke, and cardiovascular mortality). 
2. To test whether EPA+DHA supplementation reduces the risk of (a) total cancer 
(excluding non-melanoma skin cancer) and (b) major CVD events. 
Secondary aims: 
1. To test whether vitamin D3 or EPA+DHA supplementation reduces the risk of (a) incident 
colorectal cancer, (b) incident breast cancer (in women), (c) incident prostate cancer (in 
men), and (d) total cancer mortality. 
2. To test whether vitamin D3 or EPA+DHA supplementation reduces the risk of (a) an 
expanded composite cardiovascular endpoint of MI, stroke, cardiovascular mortality, and 
coronary revascularization (i.e., coronary artery bypass grafting [CABG] or percutaneous 
coronary intervention [PCI]) and (b) the individual components of the primary 
cardiovascular endpoint, particularly total CVD mortality. 
Tertiary aims: 
1. To explore whether vitamin D3 and EPA+DHA supplementation exhibit synergistic or 
additive effects on the risk of (a) total cancer, (b) major CVD events, and (c) the 
secondary endpoints specified above. 
2. To explore whether the effect of vitamin D3 or EPA+DHA supplementation on cancer and 
CVD risk varies by (a) baseline blood levels of these nutrients, (b) race/skin 
pigmentation (for vitamin D3), and (c) body mass index (BMI) (for vitamin D3). 
III. SUBJECT SELECTION 
 
Study population: VITAL will be conducted among 20,000 apparently healthy participants. 
Included in the trial will be men aged ≥60 and women aged ≥65, ages at which rates of chronic 
disease increase substantially. All participants will need to have minimally a high school 

 
4
education (to complete mail-based questionnaires). Excluded from the study will be individuals 
who have (1) a history of cancer (except non-melanoma skin cancer), MI, stroke, TIA, angina, 
CABG, or PCI; (2) have any of the following safety exclusions: use of anticoagulants at 
baseline, history of kidney stones, renal failure or dialysis, hypercalcemia, hypo- or 
hyperparathyroidism, severe liver disease (cirrhosis), or sarcoidosis or other granulomatous 
diseases such as active chronic tuberculosis or Wegener’s granulomatosis; (3) have an allergy 
to fish (for EPA+DHA); (4) have another serious illness that would preclude participation; (5) are 
consuming greater than 800 IU of vitamin D from all supplemental sources combined (individual 
vitamin D supplements, calcium+vitamin D supplements, medications with vitamin D [e.g., 
Fosamax Plus D], and multivitamins), or, and are not willing to decrease or forego such use 
during the trial (to ensure total supplemental vitamin D intake does not exceed the current safety 
limit [i.e., NOAEL] of 4000 IU/d set by the European Commission Scientific Committee on Food 
(EC SCF)); (6) are consuming greater than 1200 mg/d of calcium (the RDA for individuals aged 
>502) from all supplemental sources combined; (7) are taking fish oil supplements and are not 
willing to forego their use during the trial; and (8) are not willing to participate, as evidenced by 
not signing the informed consent form. Although thiazide diuretics and supplemental vitamin D 
may interact to increase hypercalcemia risk, it is unlikely that such an interaction would occur at 
the vitamin D dose we propose.45 Thus, thiazide use will not be an exclusion criterion. 
 
Cohort assembly: A master mailing tape containing 2.5 million names and addresses will be 
assembled by Listmart, a list broker with access to all commercially available U.S. mailing lists. 
The listings will include licensed health professionals (e.g., physicians, nurses, pharmacists, 
veterinarians, physical therapists), other professionals (e.g., executives, managers, financial 
service professionals, computer professionals, architects, engineers, accountants), AARP 
members, and subscribers to magazines that have minority readership or appeal to 
professionals and college-educated individuals (e.g., Essence, National Geographic and Time). 
Additionally, it will included individuals who have indicated interest in participating through the 
study website (www.vitaltrial.org; see paragraph below), publicized via advertisements and 
feature articles in “healthy-lifestyle” publications, as well as individuals who are currently 
participating in our other observational epidemiologic studies (e.g., Women’s Health Study). Of 
the 2.5 million names on the mailing tape, an estimated 500,000 will be black or Hispanic. Each 
invited individual will be assigned a study identification number and mailed an invitation letter 
and screening  questionnaire. 
 
In addition, we will consider a strategy of placing advertisements and feature articles in 
magazines that have minority readership and in “healthy-lifestyle” publications. The 
advertisement would explain the objectives of the trial and the general eligibility criteria. 
Interested individuals would be encouraged to visit a study web site at www.vitaltrial.org where 
they could review in greater detail the purpose of the trial, what participation involves, and the 
eligibility criteria. By submitting their name, address and age at the web site, individuals would 
be assigned a study identification number and mailed the VITAL invitation letter and a screening  
questionnaire. If necessary, we will also consider inviting eligible participants from groups with 
large minority representation, such as church groups. 
 
IV. SUBJECT ENROLLMENT 
 
Screening : The W.A. Wilde Company, a direct marketer in Holliston, MA that we have used in 
our previous trials, will mail screening  materials by non-profit bulk rate to each individual in the 
assembled cohort. The mailings will occur in groupings of approximately 500,000 each, spaced 
at monthly intervals. The mailing package will include: 

 
5
• 
A letter explaining the rationale for VITAL. The letter will outline what participation would 
entail and provide sources for further information on relevant scientific issues. We will 
also consider sending a targeted letter to individuals identified as African-American with 
a high degree of certainty (e.g., Essence subscribers). The special invitation letter would 
emphasize that African-Americans are at higher risk for vitamin D deficiency, as well as 
heart disease, stroke, and certain cancers.  
• 
A very brief screening questionnaire with items on demographics (age, gender, 
race/ethnicity, education); medical history (cancer, MI, stroke, CABG and PCI);  and 
current use   of supplements containing vitamin D or fish oil.  
• 
A self-addressed, pre-paid envelope for returning study forms. 
If the initial response rates are lower than anticipated, a second enrollment request will be 
mailed to  selected non-respondents, who will be identified from mailing groups that had high 
response and eligibility rates. 
Eligible at screening : A computer program will evaluate the screening questionnaire data to 
classify respondents’ eligibility to receive the baseline questionnaire and informed consent form 
as ineligible, eligibility pending, or eligible. Respondents with eligibility pending will be sent a 
follow-up form to clarify incomplete or inconsistent responses so that they can be classified as 
ineligible or eligible. Respondents will be eligible to receive the baseline questionnaire and 
informed consent form if they meet these criteria: (1) are age ≥60 (men) or ≥65 (women) years; 
(2) have at least a high school education (to be able to complete mail-based questionnaires); (3) 
have no history of cancer (except non-melanoma skin cancer), MI, stroke,  CABG, or PCI; (4) 
are consuming no more than 800 IU of vitamin D from all supplemental sources combined 
(individual vitamin D supplements, calcium+vitamin D supplements, medications with vitamin D 
[e.g., Fosamax Plus D], and multivitamins), or, if taking, willing to decrease or forego such use 
during the trial (to ensure total supplemental vitamin D intake does not exceed the current safety 
limit [i.e., NOAEL] of 4000 IU/d set by the EC SCF); (5) are not taking fish oil supplements, or, if 
taking, willing to forego their use during the trial.  
We estimate that approximately 125,000  respondents will be potentially  eligible  for 
participation in the VITAL trial based upon their responses on the screening questionnaire. 
Respondents who are not eligible will be mailed a letter thanking them for their interest and 
informing them of the general criteria that determined eligibility. 
Enrollment: Every two weeks, the names/addresses of recent respondents, who are eligible to 
receive the baseline questionnaire, will be electronically compiled in preparation for the mailing 
of the baseline questionnaire and informed consent form. It is anticipated that there will be 5-
10,000 entries per bi-weekly list. W.A. Wilde Company will mail these materials by non-profit 
bulk rate to each: 
• 
A letter of appreciation for the initial response which also reiterates the purpose of the 
study and what participation involves.  
• 
An informed consent form. 
• 
A baseline questionnaire with items on demographics (date of birth); medical history 
(kidney stones, hypercalcemia, kidney failure, sarcoidosis, liver disease, thyroid disease, 
chronic tuberculosis, other major illnesses); allergy to fish; current use of calcium 
supplements and other supplements containing vitamin D or fish oil; height and weight; 
use of anticoagulant medications; and potential effect modifiers such as skin 
pigmentation and sunlight exposure. Respondents will also be asked for telephone 

 
6
number(s) and an e-mail address in the event that follow-up needs to be conducted in 
order to clarify questionnaire responses and to provide their social security number 
(optional) which will be used for identification purposes only. In addition, the form will 
contain a question asking whether individuals would be willing to return a blood sample if 
mailed a convenient blood collection kit. 
• 
A self-addressed, pre-paid envelope for returning study forms. 
Eligibility: A computer program will evaluate the questionnaire data to classify respondents’ 
eligibility as ineligible (to enter the run-in), eligibility pending, or eligible. The “eligibility pending” 
respondents will be sent a follow-up form to clarify incomplete or inconsistent responses. 
Respondents will be eligible if they meet these criteria: (1) have none of the following safety 
exclusions: use of anticoagulants, history of kidney stones, renal failure or dialysis, 
hypercalcemia, hypo- or hyperparathyroidism, severe liver disease (cirrhosis), or sarcoidosis or 
other granulomatous diseases such as active chronic tuberculosis or Wegener’s 
granulomatosis; (2) have no allergy to fish (for EPA+DHA) or fish oil supplements; (3) have no 
other serious illness that would preclude participation; (4) are consuming no more than 800 IU 
of vitamin D from all supplemental sources combined (individual vitamin D supplements, 
calcium+vitamin D supplements, medications with vitamin D [e.g., Fosamax Plus D], and 
multivitamins), or, if taking, willing to decrease or forego such use during the trial (to ensure total 
supplemental vitamin D intake does not exceed the current safety limit [i.e., NOAEL] of 4000 
IU/d set by the EC SCF); (5) are consuming no more than 1200 mg/d of calcium (the RDA for 
individuals aged >502) from all supplemental sources combined; (6) are willing to participate, as 
evidenced by signing the informed consent form.  
We estimate that approximately 40,000 individuals will be both willing and fully eligible to 
participate in the trial and enter the run-in. Respondents who are not eligible will be mailed a 
letter thanking them for their interest and informing them of the general criteria that determined 
eligibility. 
Run-in: The experience of other studies conducted by our research group (PHS, WHS, and 
WAFACS) has demonstrated the success of a run-in period in identifying a group of excellent 
compliers for long-term follow-up. Because the health experience of participants will be 
analyzed according to randomized treatment assignment, the use of a run-in phase to eliminate 
poor compliers prior to randomization will increase the power of the study.46 During a 3-month 
run-in period, all participants will take placebo vitamin D and placebo fish oil. As the effects of 
both agents on cancer prevention are likely to be chronic, it would not be scientifically 
appropriate to use active agent in the run-in and then randomize to placebo. Moreover, the use 
of placebos will allow us to most clearly analyze the side effects of the study agents during the 
randomized treatment period.  
At trial enrollment, willing and eligible participants will be mailed a 6-month supply of run-in 
calendar packs (i.e., 3 months for the actual run-in and 3 months to allow pill-taking to continue 
uninterrupted while we check participants’ responses for continued eligibility and then ship 
randomized pills to participants who remain eligible). Participants will take two  pills per day (one 
vitamin D placebo pill and one  fish oil placebo capsule). At 3 months, participants will receive a 
follow-up run-in questionnaire asking about continued eligibility, willingness to continue in the 
trial, pill compliance, and potential side effects. Non-responders will be mailed second and third 
requests at 6-week intervals.  

 
7
Participants will be randomized only if they: (1) demonstrate good compliance in pill taking, 
defined as taking at least two-thirds of the study pills during the run-in; (2) express willingness to 
continue in the trial; (3) report no new history of cancer (except non-melanoma skin cancer), MI, 
stroke, TIA, angina, CABG, PCI, use of anticoagulants, kidney stones, hypercalcemia, 
sarcoidosis, or other serious illness during the run-in; (4) are consuming no more than 1200 
mg/d of calcium (the RDA for individuals aged >502) from all supplemental sources combined; 
and (5) demonstrate continued willingness to limit supplemental vitamin D intake to ≤800 IU/d 
and to forego the use of fish oil supplements. 
We estimate that of the 40,000 initially willing and eligible individuals enrolled in the run-in, 50% 
(n=20,000) will be compliant and remain willing and eligible for randomization.  
Randomization: Willing and eligible participants will be randomly assigned to one of four 
treatment groups: vitamin D3 (2000 IU/d) and fish oil (EPA+DHA, 840  mg/d); vitamin D3 and fish 
oil placebo; placebo vitamin D3 and fish oil; and placebo vitamin D3 and placebo fish oil. 
Individuals will be randomized to treatment within 5-year age groups using a computer-
generated table of random numbers. Within each age group, treatment assignments will be 
generated in blocks of eight individuals, with two individuals in each of the four treatment 
combinations. The date of randomized treatment assignment will be the formal “in-study” date. 
(The use of age stratification during randomization will increase statistical efficiency and power.) 
Participants will be mailed an annual supply of pills ( in  calendar packs) and instructed to take 
two pills – one of vitamin D or placebo and one  of fish oil or placebo – each day.  
V. STUDY PROCEDURES 
A. BLOOD COLLECTION  
Baseline: Participants who indicate on their baseline questionnaire a willingness to provide an 
optional venous blood sample will be sent a blood collection kit during the run-in. Participants 
who indicate on the questionnaire that they are “not sure” about participating in the blood 
collection, or who do not answer the question regarding participation, will be called to clarify 
their degree of interest. The kit will include a consent form, supplies and instructions for having 
the blood drawn into two EDTA tubes, one heparin tube, and one citrate tube, a gel-filled freezer 
pack, and an overnight courier air bill. We anticipate that most participants will have their blood 
drawn by their own healthcare providers or in another healthcare or blood-drawing facility. 
Participants will be instructed to provide a fasting sample, if possible, and to record the time of 
venipuncture and the time of their last meal. They will be instructed to send their sample to our 
blood laboratory in the freezer packs within 24 hours of the blood draw. Upon receipt, the 
samples will be centrifuged to separate plasma, red blood cells, and buffy coat; aliquoted into 2-
ml Nunc vials (including multiple vials of EDTA, heparin, and citrate plasma, and  vials  for 
EDTA red blood cells, and  buffy coat and stored in individual vial locations in nitrogen freezers(-
170 ºC). The process will be completed within several hours of specimen receipt to ensure that 
samples are frozen within 30-36 hours after venipuncture. Each freezer has an electronic alarm 
which is monitored 24 hours a day. If problems arise, staff members are paged by the alarm 
company and can adjust the levels of liquid nitrogen as needed. This system has protected 
stored samples from inadvertent thawing or warming for nearly 20 years.  
The main reason for collecting pre-randomization blood specimens is to provide the opportunity 
to assess whether treatment effects are modified by baseline blood levels of 25(OH)D and 
EPA+DHA. When stratifying by 25(OH)D, season (assessed by date of return) and geographic 

 
8
location (assessed by zip code) will be considered. This resource will also be extremely 
valuable for future applications for funding of ancillary studies to explore other genetic and 
biochemical hypotheses in this well-characterized cohort. A nested case-control or case-cohort 
approach can be used, enabling the study of many promising biochemical and genetic markers 
(e.g., VDR polymorphisms) as potential predictors of disease risk and modifiers of intervention 
effects at very low incremental cost. 
Follow-up: A follow-up fasting blood sample will be collected in year 2 and year 4  from a 
randomly selected subset of 2000 participants who provided a baseline fasting blood sample. 
These follow-up samples, to be collected using the same methods as for the baseline samples, 
will allow assessment of (a) pill-taking compliance, (b) changes in biomarkers with treatment, 
and (c) in the placebo group, the effect of changing trends in background fortification with 
vitamin D and omega-3 fats. The samples will be particularly important for an exploratory 
analysis of treatment-induced changes in 25(OH)D levels among black participants, as there are 
few data on this topic.47 In addition, changes in blood calcium and parathyroid hormone (PTH) 
levels will be examined as markers of possible hypercalcemia, a potential side effect of high 
vitamin D intake. The bloods will also be archived for future ancillary studies to evaluate whether 
treatment with one or both of the study agents leads to changes in biochemical factors, 
including those in the lipid, glucose tolerance, inflammation, endothelial dysfunction, thrombosis, 
insulin, and insulin-like growth factor pathways. 
Biochemical assays: Blood levels of 25(OH)D and EPA+DHA will be assayed in the final 2 
years of the study in a case-cohort design on a subset of participants who provide an 
analyzable sample at baseline and all 2000 participants who provide a blood sample at follow-
up. In addition, baseline and follow-up blood levels of calcium and PTH will be assayed in the 
latter group. The 25(OH)D assay will be performed by the laboratory of Dr. Bruce Hollis at the 
Medical University of South Carolina in Charleston (a consultant), and the other assays will be 
performed by the laboratory of Dr. Nader Rifai at Children’s Hospital in Boston, MA.  
The sample for analysis of baseline blood levels will be selected to maximize efficiency within 
budgetary constraints. A case-cohort design,48 which has efficiency advantages over case-
control studies, will be employed to minimize cost while allowing the unbiased estimation of 
hazard ratios as well as absolute risk for individuals. The common subcohort sample can be 
used as a reference risk set for more than one outcome—total cancer and total CVD in this 
proposal. Over 5 years of follow-up, we would expect to accrue approximately 1660 cancer 
cases and 990 CVD cases, with somewhat smaller numbers if the agents are highly effective. If 
60% of participants provide blood specimens, this would include about 996 cancer cases and 
594 CVD cases with bloods. The size of the subcohort sample will be initially set at 2410, to 
reach a total of 4000 blood assays and provide more than two non-cases per case for each 
outcome. However, 12-13% of the subcohort will likely be in one of the case groups. Thus, we 
will resample from the main cohort to increase the subcohort size by the number of duplicates to 
maintain the total number of assays at 4000. Control selection will be stratified by gender and 
baseline age (within 5-year groups) to frequency-match the distribution in the total case group.  
B. ASSESSMENT OF DIETARY AND SUPPLEMENTAL INTAKE 
 
A validated, self-administered semi-quantitative food frequency questionnaire (FFQ) will be 
mailed to participants during the run-in. This questionnaire, developed by Walter Willett, MD, 
DrPH, and colleagues, is an efficient, reliable, and accurate instrument for categorizing 
individuals according to their intake of 32 nutrients, including vitamin D and marine omega-3 

 
9
fats.49-54 Participants will be asked to estimate their average intake over the past year of various 
foods, beverages, and supplements that contain vitamin D, marine omega-3 fats, and other 
nutrients. Dietary vitamin D intake will be estimated from participants’ reported intakes of certain 
foods, including dairy products, fortified breakfast cereals, fortified juices, dark-meat fish, and 
cod liver oil. Dietary marine omega-3 fat intake will be estimated from intake of dark-meat fish, 
canned tuna, other fish, and seafood. Classifying participants by baseline intake of various 
nutrients will allow us to evaluate whether the study agents’ effects vary by such intake. 
Participants will also be asked to complete the FFQ at 2 years and at trial’s end, enabling an 
examination of dietary changes over time. 
Questions on use of non-study supplements or drugs containing vitamin D or EPA+DHA will be 
asked at baseline, 6 months, and on yearly follow-up questionnaires. We will also assess 
flaxseed and alpha-linolenic acid intakes. We will ascertain and analyze intake from food and 
supplements separately, and from both sources combined.  
C. TREATMENT 
In our research application, we proposed testing 1600 IU (40 μg) per day of vitamin D3 
(cholecalciferol) in VITAL. Since the application was submitted, based on new research and with 
approval from NCI, we now will test 2000 IU (50 µg) per day instead. We seek to obtain a large-
enough difference in vitamin D status between the treatment and placebo groups to detect 
health benefits, and there appear to be few safety issues associated with this dose. Recent 
studies have found little toxicity risk with doses up to 10,000 IU/d.45 (Nevertheless, to further 
ensure participants’ safety, we will exclude from the trial persons with a history of kidney stones, 
hypercalcemia, or sarcoidosis [Section 5.2]; follow-up calcium and parathyroid hormone [PTH] 
levels will be obtained in a random subsample of participants [Section 5.4].) Accumulating 
evidence suggests that vitamin D intakes well above currently recommended amounts of 400 
IU/d for adults aged 50-70 and 600 IU/d for adults aged >702 are necessary for health benefits. 
In a review of studies of serum 25(OH)D in relation to bone mineral density, lower-extremity 
function, dental health, and risk of falls, fractures, and colorectal cancer, Bischoff-Ferrari et al.56 
found that, for all endpoints, advantageous 25(OH)D levels began at 75 nmol/L, and optimal 
levels were between 90-100 nmol/L. The average older individual requires an oral vitamin D3 
intake of at least 800-1000 IU/d (20-25 µg) to achieve a serum 25(OH)D of 75 nmol/L.57 Among 
postmenopausal women in the WHI, 400 IU/d of vitamin D3 raised median plasma 25(OH)D 
from 42.3 to only 54.1 nmol/L.44, 58 
The primary reason for the dose change is a recent study by Aloia et al59 which indicates that 
the dose-response for serum 25(OH)D with vitamin D intake is not linear, but that the rate of 
increase in serum levels decreases at higher levels of intake. At a dose of 800 IU (20 μg/day), 
the rate of change is 1.2 nmol/L.  However, at doses ≥1,400 IU (35 μg/day), the rate of change 
decreases to 0.7 nmol/L.  Thus, extrapolation of these data suggests that with the originally 
proposed 1600 IU/d (40 μg/day), the expected change in mean serum 25(OH)D levels would be 
only 28 nmol/L, or a change from an estimated 42 nmol/L at baseline to a final mean level of 70 
nmol/L.  With 2000 IU/d (50 μg/day), the increase would be 35 nmol/L from baseline to a final 
mean level 77 nmol/L, which would be above the 75 nmol/L threshold level at which health 
benefits are believed to occur 56 , as well as a level believed to be free of any risks. 
 
Potential side effects of vitamin D are rare.  They include gastrointestinal (GI) upset (presence 
or absence of symptoms of peptic ulcer, nausea, constipation, diarrhea), and physician 
diagnosis of hypercalcemia or kidney stones.  To minimize the risks of hypercalcemia and 

 
10
kidney stones, we will require that participants limit their total intake of calcium from all sources, 
including multivitamins, single supplements of calcium, and other drugs that contain calcium to 
1200 mg or less a day, 
Active vitamin D3 (2000 IU [50 µg] per pill) and matching inert placebo will be provided by 
Pharmavite LLC.  
 
In our research application, we proposed testing a total EPA+DHA dose of 1 mg/d (EPA [500 
mg/d] + DHA [500 mg/d]). Since the application was submitted, we have worked with various 
drug companies to develop soft-gel capsules containing these doses. We now propose to test a 
total of EPA+DHA dose of 840 mg/d instead, since this is the highest dose of EPA + DHA 
currently available in a 1000 mg capsule. 
We believe that this dose provides the best balance of feasibility, efficacy and safety. We seek 
to obtain a large-enough difference in omega-3 fatty acid status between the treatment and 
placebo groups to detect health benefits, and there appear to be few safety issues associated 
with this dose. Health authorities recommend 400 mg to 1 g/d for cardioprotection.60 A total 
dose of 850 mg/d was used in the GISSI-Prevenzione Trial (EPA to DHA ratio, 1:2) and AREDS 
2 (EPA to DHA ratio, 2:1), while a dose of 1.8 g/d of EPA was used in JELIS. For this trial, we 
have selected a total dose of omega-3 fatty acids recommended by the American Heart 
Association for cardioprotection and demonstrated to be beneficial in one secondary prevention 
population with minimal side effects.42 Because the optimal ratio of EPA to DHA is unknown,61, 62 
we have selected a 40:30 ratio of EPA to DHA. The ratio of omega-3 to omega-6 fatty acid 
intake is between 1:10 and 1:20 in most Western countries, including the U.S., whereas the 
optimal ratio for disease prevention has been hypothesized to be closer to 1:1 or 1:2,63, 64 
although this is controversial.65 Indeed, there is growing consensus that the absolute intake of 
omega-3 is a more important predictor of health than is the ratio of omega-3 to omega-6 intake, 
at least for cardiovascular outcomes.65-68 However, given that the average intake of EPA+DHA 
is 100-200 mg/d among U.S. adults,60 the proposed intervention of 840 mg/d would be expected 
to increase the average participant’s omega-3 intake by a factor of 4 to 8. Assuming no 
concurrent change in omega-6 intake, the proposed omega-3 dose would thus have the effect 
of achieving the purported optimal omega-3 to omega-6 ratio and providing intakes associated 
with benefits in previous studies. 
As with vitamin D, potential side effects of omega-3 fatty acids are rare.  They include 
gastrointestinal (GI) symptoms (stomach upset or pain, nausea, constipation, diarrhea), 
bleeding (any GI bleed, GI bleeding requiring transfusion, hematuria, easy bruising, epistaxis), 
skin rash, colds or upper respiratory tract infection, flu-like symptoms, bad taste in mouth, and 
physician diagnosis of atrial fibrillation or other irregular rhythms.  The FDA has concluded that 
marine omega-3 fatty acid doses of up to 3 g/d are “Generally Recognized as Safe.”69  The AHA 
also has concluded that these risks are very low or low at doses of up to 1 g/d and low to 
moderate at doses of 1-3 g/d.12  Because they undergo an extensive purification process, high-
quality fish oil supplements are free of environmental toxins (e.g., methylmercury, 
polychlorinated biphenyls [PCBs], and dioxins) found in some fish.12, 71  
Active fish oil (1000 mg capsule containing 840 mg EPA + DHA ) and matching inert placebo 
will be provided by Pronova BioPharma ASA of Norway.  
 

 
11
D. FOLLOW-UP 
With regard to follow-up, at 6 months, participants will be mailed a very brief follow-up 
questionnaire and at each anniversary date a more detailed questionnaire and a re-supply of 
calendar packs. The questionnaire will include items on compliance with randomized 
treatments, use of non-trial supplements of vitamin D and fish oil, use of calcium supplements, 
development of major illnesses, dietary intakes of vitamin D and fish, and updated risk factors 
related to cancer and CVD. Information on potential side effects of the study agents will be 
elicited for monitoring by the Data and Safety Monitoring Board. For fish oil, these side effects 
include GI symptoms (stomach upset or pain, nausea, constipation, diarrhea), bleeding (any GI 
bleed, GI bleeding, hematuria, easy bruising, epistaxis), skin rash, colds or upper respiratory 
tract infection, flu-like symptoms, bad taste in mouth, and physician diagnosis of atrial fibrillation 
or other irregular rhythms. For vitamin D, these side effects include GI symptoms as listed 
above, and physician diagnosis of hypercalcemia or kidney stones. Non-responders will be 
mailed a second and third request and will then be telephoned to collect study data. At the very 
least, vital status will be ascertained. Participants will be instructed to discontinue their study 
pills if, during follow-up, they receive a diagnosis of kidney stones, hypercalcemia, sarcoidosis, 
or other safety-exclusion conditions. Likewise, participants will be advised to discontinue the 
vitamin D component if, in the course of follow-up, they report taking supplementary vitamin D 
(not from diet) that amounts to greater than 800 IU per day or daily calcium supplements that 
amount to greater than 1200 mg per day. 
At 6-month intervals between annual follow-ups, participants will be mailed an interim form with 
questions on the development of primary endpoints (cancer, MI, and stroke), difficulties with pill 
compliance, and address changes. Participants will be asked to return the form only if one or 
more questions apply. A newsletter will be included with each interim mailing to keep 
participants abreast of the trial’s progress and provide information on related health topics. The 
interim follow-up will help the staff maintain accurate contact information and provide a head 
start in addressing compliance issues and timely collection of medical records to confirm 
endpoints.  
E. COMPLIANCE 
Our primary measure of compliance will be the self-reported information provided on the yearly 
follow-up questionnaires, which will ask about adherence to the pill-taking regimen. Our 
experience in PHS, WHS and WAFACS indicates that although most participants try diligently to 
adhere to the regimen, those who do not comply have no embarrassment about describing what 
they are actually doing. Thus, blood levels have shown near-perfect correlations with self-
reported questionnaire data on adherence.72 Because VITAL participants will reside throughout 
the U.S., it will not be possible to obtain blood samples for a validity study on all, or even a 
random sample of, participants. Thus, at 6-months and thereafter every  year we will visit 
unannounced 100  New England-area participants, to request an on-the-spot blood sample to 
be analyzed for 25(OH)D and EPA+DHA levels. The distribution of these values will be 
compared between the active and placebo groups, and compared with the questionnaire data 
on compliance, as a test for validity. Individuals contacted in this manner will be asked to sign 
an informed consent for the blood draw. Additionally, we will be able to assess 25 (OH)D and 
EPA + DHA levels in the 2000 randomly selected participants who will be asked to provide a 
blood sample at year 2 and year 4, according to active and placebo groups. 

 
12
F. ENDPOINT ASCERTAINMENT AND VALIDATION 
The primary cancer endpoint of VITAL will be total cancer incidence (excluding non-melanoma 
skin cancer), with cancer mortality and the individual sites of colorectal, breast, and prostate 
cancer as secondary endpoints. For CVD, the primary endpoint will be a composite endpoint of 
MI, stroke, and CVD mortality. Secondary endpoints will include a second composite endpoint 
adding revascularization procedures of CABG and PCI, as well as the individual endpoints of 
MI, stroke, revascularization, and CVD mortality.  
Participants who self-report one of the above study endpoints will be asked to sign a medical 
release form authorizing VITAL staff to obtain hospital/physician records. The request will be 
accompanied by a cover letter expressing sympathy for the diagnosis and explaining HIPAA 
guidelines and the scientific importance of record validation. Non-responders will be sent two 
additional requests for the medical release form. After the release is obtained, a copy will be 
sent to the treating hospital/physician. If there is no response within 1 month, a second request 
will be mailed, followed by a phone call.  
After all records are obtained, an Endpoints Committee of physicians, blinded to the randomized 
treatment assignment, will review the file and, using a defined protocol, will confirm or 
disconfirm the case. A cancer diagnosis will be confirmed with histologic or cytologic evidence. 
In the absence of these diagnostic tests, strong clinical evidence accompanied by radiologic 
evidence or laboratory markers (e.g., PSA levels) will be used to confirm cancer occurrence. 
The histologic type, grade, and stage of cancer will be recorded.73 MI will be confirmed using 
Joint European Society of Cardiology/American College of Cardiology Foundation/ American 
Heart Association/World Heart Federation (ESC/ACCF/AHA/WHF) Task Force for the 
Redefinition of Myocardial Infarction criteria.74 Stroke will be confirmed and categorized 
according to Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria.75 Death due to a 
cardiovascular cause will be confirmed by convincing evidence of a CVD event from all 
available sources, including death certificates, hospital records, autopsy, and observer accounts 
(for deaths outside the hospital). 
For reported deaths, the validation process will be similar. A condolence letter will be mailed to 
the family, requesting permission to obtain medical records and a copy of the death certificate. If 
the family does not provide the death certificate, a copy will be requested from the state vital 
records bureau where the participant died. The Endpoints Committee will review all records 
relevant to the death and assign an ICD code. At the end of the trial, we will search the National 
Death Index Plus (NDI-Plus) data base for known deaths for cases where we could not obtain 
records. NDI-Plus provides an ICD-coded cause of death based on death certificate information. 
We will also use the NDI to search for participants who become lost to follow-up. 
G. DATA MANAGEMENT 
Because participants will be followed solely by mail, our computing system is a critical feature of 
effective follow-up. This system, which was developed and fine-tuned in our previous trials, will 
track each participant’s stage in the study and level of participation. It will automatically generate 
letters, questionnaires, and phone call reminders at the appropriate times. Names, addresses, 
telephone numbers, participation status, and processing information will be kept up to date, and 
data from questionnaires, letters, and phone calls will be entered into the study database. When 
talking to a participant, study personnel will need ready access to identifying information, 
participation level, and the content of previous study-related telephone calls. However, it is also 

 
13
critical that these data be available only to authorized staff members. Our existing computer and 
security systems, which balance these considerations, will be used in VITAL.  
Questionnaire data will be optically scanned into the computer. The relevant software—
TELEform and Alchemy (Cardiff Software)—has been successfully used for several years in the 
WHS and PHS. Out-of-range, internally inconsistent, and unclear data will be reviewed by a 
verifier who will edit misread variables. Forms that cannot be scanned, and name and address 
changes, will be entered using traditional double-entry procedures. All data will undergo 
additional within-form and across-time checks to verify accuracy. The database will be 
maintained on a UNIX server. All data files will be backed up nightly, ensuring at least two 
current copies at all times. Each month, a set of data files will be taken off-site for long-term 
storage. 
Participants’ names and contact information will be accessible only to staff members who need 
the information for their jobs. Endpoint and health-related questionnaire data will be stored in 
separate files from the processing data and will be accessible only to approved investigators 
and programmers. In these files, participants are identified only by study ID. 
VI. 
BIOSTATISTICAL ANALYSIS 
A. DATA ANALYSIS  
Analyses of treatment effects will be based on the intent-to-treat principle. The first analysis will 
compare baseline characteristics by randomized treatment assignment to ensure that balance 
was achieved by the randomization. Characteristics to be examined include known risk factors 
for cancer and CVD, including age; gender; race/ethnicity; BMI; smoking; alcohol use; physical 
activity; medical conditions such as hypertension, hyperlipidemia, diabetes, and family history of 
cancer and CVD; and baseline vitamin D and omega-3 fatty acid levels as assessed by dietary 
questionnaire in all participants and blood assays in a case-cohort subsample. The large 
sample size, as well as successful balance of known potential confounders, will provide 
assurance that unmeasured or unknown potential confounders will also be equally distributed 
across randomized treatment groups. 
In this 2x2 factorial design, the primary aim is to compare the main effects of intention-to-treat 
with vitamin D and with fish oil on cancer and CVD. We will use the Cox proportional hazards 
model to allow for variable follow-up lengths76 and will estimate the hazard ratio for each 
intervention using indicators for treatment exposure, controlling for the second intervention, age, 
and gender. Because the cohort will consist of older individuals, competing risks due to deaths 
from other causes will be considered. The primary analysis will estimate the cause-specific 
hazard and the hazard ratio comparing intervention groups for each outcome of interest by 
censoring individuals with deaths due to competing causes. To estimate the cumulative 
incidence function, the subdistribution of each endpoint will be plotted over time.77, 78 The 
alternative Fine and Gray approach79 models the effect of treatment on the subdistribution 
hazard or directly on the cumulative incidence function. While we will consider this and compare 
its fit to the model-free cumulative incidence curves,78 the proportional hazards approach will be 
our primary analysis. 
Similar analyses will be conducted for both cancer and CVD. For cancer, we will estimate the 
intervention effects on the primary endpoint of total cancer, as well as the secondary outcomes 
of breast, prostate, and colorectal cancer, and cancer mortality. For CVD we will estimate the 

 
14
effects on the primary composite endpoint of major CVD (MI, stroke, and cardiovascular 
mortality); on the secondary composite endpoint that additionally includes coronary 
revascularization; and on the individual components of MI, stroke, revascularization, and 
cardiovascular mortality. 
Beyond the primary analyses, we will examine effect modification by the other randomized 
intervention, by baseline risk factors, and by time. We have a particular interest in exploring 
interactions between the interventions, as well as with the baseline plasma biomarkers of 
25(OH)D for vitamin D and of EPA+DHA for fish oil. We hypothesize that the intervention effects 
may be larger among those with below-median baseline levels, and will examine treatment 
effects by quartiles of these biomarkers. The biomarker data will be analyzed as a case-cohort 
study using methods for proportional hazards regression.80 Because of the frequency matching 
by age and gender, appropriate stratum-specific weighting of the observations will be 
employed.81 We also have a prior interest in the effects of the vitamin D intervention within 
groups defined by race/ethnicity and skin pigmentation, and by BMI. In addition, we will evaluate 
effect modification by age and gender, as well as by sunlight exposure, calcium and phosphorus 
intakes from the FFQ (as these nutrients affect vitamin D bioavailability82), and baseline risk 
factors for cancer and CVD. The latter interaction effects will be interpreted cautiously, as 
hypothesis generating. Finally, we will examine whether treatment effects vary over time and 
duration of treatment by examining survival plots and interactions with time. There may be a 
latent effect on cancer incidence, depending on the stage of carcinogenesis during which these 
agents act. 
We will also compare the incidence of potential side effects in the active v. placebo groups for 
each agent, including the incidence of kidney stones with vitamin D assignment (cf. WHI 
calcium-vitamin D findings44) and the incidence of GI symptoms, skin abnormalities, and 
bleeding with fish oil assignment (cf. JELIS findings41). 
B. POWER CALCULATIONS  
To calculate power, the following assumptions were made: (1) a 2x2 factorial trial in 10,000 men 
aged >60 and 10,000 women aged >65; (2) independent and equal allocation of participants to 
each treatment; (3) an age distribution based on that observed at baseline in our past trials for 
men aged ≥60 and women aged ≥65; (4) age-specific event rates based on the observed rates 
in the first 5 years of the PHS II for men and the WHS for women; (5) trial follow-up of 5 years, 
with little loss to follow-up as achieved in our past trials; and (6) compliance similar to that in 
published trials upon which our estimated rate ratio (RR) reductions are based. The cited 
reductions are thus the observed effects we would see in the trial. The corresponding ‘true’ RR 
is given assuming an average compliance of 80%. Power is given for a two-sided test using a 
logrank analysis83 with a significance level of 0.05. 
Cancer: Assuming that 
only one agent is 
effective, with 5 years of 
treatment and follow-up 
we will have 91% power 
to detect an observed 
RR of 0.85 for the 
primary endpoint of total 
cancer incidence (Table 
Table 1. Power for effects of a single agent on cancer in a factorial trial of 10,000 men 
aged≥60 and 10,000 women aged≥65 with 5 years of follow-up 
Observed 
RR† 
True 
RR 
Total   
Cancer 
Cancer 
Mortality 
Colorectal 
Cancer 
Breast 
Cancer 
(Women) 
Prostate 
Cancer 
(Men) 
0.90 
0.875 
58.7 
- 
- 
- 
- 
0.85 
0.812 
91.1 
- 
- 
- 
- 
0.80 
0.750 
99.4 
52.0 
31.8 
34.6 
70.1 
0.75 
0.687 
>99.9 
72.2 
47.0 
50.9 
88.3 
0.70 
0.625 
>99.9 
87.3 
63.1 
67.5 
96.9 
0.65 
0.560 
>99.9 
95.6 
77.6 
81.5 
99.5 
0.60 
0.500 
>99.9 
98.9 
88.4 
91.2 
>99.9 
†Observed RR=intent-to-treat RR, including noncompliant participants. Compliance assumed 
to be 80%. True RR=that with perfect compliance. 

 
15
1). For prostate cancer, we will have 88% power to detect a 25% risk reduction. For breast 
cancer, we will have 81% power to detect a 35% risk reduction. For colorectal cancer, we will 
have 78% power to detect a 35% risk reduction and 88% power to detect a 40% risk reduction. 
For cancer mortality, we will have 87% power to detect a 30% risk reduction. If both agents are 
effective in preventing cancer but act independently, power would be reduced slightly due to a 
somewhat smaller overall number of events. If the agents interact, however, power will be 
affected to the extent of the interaction. Should the agents act synergistically, power would 
increase, as illustrated in Table 2 for the outcome of total cancer.  
For example, if the effect of each agent alone is a reduction of 10%, but in combination the 
effect is stronger, with an additional 10% decrease, the RR comparing the combined group to 
the all placebo group would be 0.73, versus 0.81 with additive effects (on the multiplicative 
scale). Power for the main effect of each agent would then increase to 86% (or higher with 
greater synergy). The trial would have power to detect moderate-to-large interactions, as shown 
in Table 2. Should the agents interact in a subadditive fashion, power would be reduced to the 
extent of the subadditivity. Power to explore effect modification by baseline blood levels of 
25(OH)D and EPA+DHA among those above and below the median level in the cohort will be 
lower because we anticipate that about 60% of participants will provide blood specimens. We 
will have about 90% power to detect an interaction of size 0.6. 
Power has been shown for a given observed reduction in risk, assuming compliance similar to 
that seen in current randomized trials such as the WHS and PHS. For total cancer, our power 
will be excellent with a 5-year follow-up even if compliance is lower. For example, if the true 
reduction in risk is 20%, the observed RR corresponding to compliance of 80% is 84%, and we 
have 94% power to detect this level of effect. If compliance is only 75%, the observed RR would 
be 0.85, for which we would have 91% power. Finally, if compliance is only 70%, the observed 
RR would be 0.86, and we would still have 87% power to detect an effect of this magnitude. 
 
 
 
 
 
 
 
 
 
CVD: As shown in Table 3, assuming that only one agent is effective, with 5 years of treatment 
and follow-up we will have 92% power to detect an observed RR of 0.80 for the primary 
composite endpoint of MI, stroke, and cardiovascular mortality. For the secondary composite 
Table 2. Power for interaction effects on total cancer in a factorial trial of 
10,000 men aged ≥60 and 10,000 women aged ≥65 with 5 years of follow-up 
RR 
Single agent† 
Interaction† 
RR  
Both agents 
Power 
Main Effect      Interaction 
0.90 
1.0 
0.81 
56.3 
 
 
0.9 
0.73 
86.3 
 
 
0.8 
0.65 
98.0 
57.9 
 
0.7 
0.57 
99.9 
92.1 
0.85 
1.0 
0.72 
88.7 
 
 
0.9 
0.65 
98.1 
 
 
0.8 
0.58 
99.8 
55.0 
 
0.7 
0.51 
>99.9 
90.2 
0.80 
1.0 
0.64 
98.8 
 
 
0.9 
0.58 
99.9 
 
 
0.8 
0.51 
>99.9 
52.0 
 
0.7 
0.45 
>99.9 
87.9 
†RR = intent-to-treat RR, including noncompliant participants (assuming 80% 
compliance). Represents the effect among those not assigned to the other 
intervention and assumes the same effect for both agents. The interaction is the 
RR for the combined group divided by the product of risks for the two separate 
groups—i.e., RRint=RRboth/(RR vit D alone* RR fish oil alone). An interaction=1 
implies additive effects (no interaction). 

 
16
endpoint of MI, stroke, cardiovascular mortality, and coronary revascularization, we will have 
99% power to detect a 20% reduction and 90% power to detect a 15% reduction. With a RR of 
0.70 we will have 87% power for cardiovascular mortality, 88% power for MI, and 90% power for 
stroke. If 
both agents are 
effective in 
preventing CVD 
but act 
independently, 
power 
would be 
reduced 
only slightly due 
to a 
smaller overall 
number of 
events. If the 
agents act 
synergistically, 
power 
would increase, 
as 
described for 
cancer. If 
the agents 
interact in subadditive fashion, power would be reduced to the extent of the subadditivity.  
 
 
 
 
 
 
For total CVD, our power would be somewhat lower—but still adequate—with more limited 
compliance. If the true effect is a 25% reduction, power would be 92% if compliance is 80% 
(observed RR=0.80), 89% if compliance is 75% (observed RR=0.813), and 84% if compliance is 
70% (observed RR=0.825). To achieve 80% power for CVD, we would need an observed RR of 
at least 0.83, which corresponds to a true reduction of 21% under 80% compliance and 24% 
under 70% compliance. 
 
VII. RISKS AND DISCOMFORTS 
 
The VITAL trial will test a dose of 2000 IU (50 µg) per day of vitamin D3 (cholecalciferol) and 840 
mg/d of EPA+DHA in VITAL. These doses provide the best balance of efficacy and safety, 
based on careful review of the literature and extensive consultation with numerous nutritional 
experts. We seek to obtain an adequate difference in vitamin D and EPA+DHA levels between 
the treatment and placebo groups to detect health benefits, and there appear to be few safety 
issues associated with these doses. For vitamin D, the selected dose is at the tolerable upper 
intake level (UL) for vitamin D3 (50 µg, or 2000 IU) set by the Food and Nutrition Board of the 
Institute of Medicine (IOM) and the European Commission Scientific Committee on Food (EC 
SCF).  Also, because we will exclude from the trial persons who report supplemental vitamin D 
intakes >800 IU/d, the selected dose ensures that no participants will be taking a supplemental 
vitamin D dose above 2800 IU, which is the no-observed-adverse-effect level (well below the 
4000 IU NOAEL specified by the EC SCF. To further ensure participants’ safety, we will exclude 
Table 3. Power for effects of a single agent on CVD in a factorial trial of 10,000 men 
aged ≥60 and 10,000 women aged ≥65, with 5 years of follow-up 
Observed 
RR† 
True 
RR 
Major 
CVD 
Total 
CVD 
CVD 
Mortality 
MI 
Stroke 
0.90 
0.875 
38.1 
57.3 
- 
- 
- 
0.85 
0.812 
71.1 
90.2 
- 
- 
- 
0.80 
0.750 
92.5 
99.2 
51.8 
53.5 
56.1 
0.75 
0.687 
99.0 
>99.9 
71.9 
73.8 
76.3 
0.70 
0.625 
99.9 
>99.9 
87.1 
88.5 
90.3 
0.65 
0.560 
>99.9 
>99.9 
95.6 
96.3 
97.1 
0.60 
0.500 
>99.9 
>99.9 
98.9 
99.1 
99.4 
†Observed RR=intent-to-treat RR, including noncompliant participants. Compliance is 
assumed to be 80%. True RR=that with perfect compliance. 

 
17
from the trial persons with a history of kidney stones, hypercalcemia, renal failure, cirrhosis, or 
sarcoidosis or other granulomatous disease (Section 5.2), and will obtain calcium and PTH 
levels in a random subsample of participants. The dose of fish oil used in this trial (840 mg/day) 
is consistent with guidelines set by health authorities who recommend up to 1 gram/day for 
cardio-protection. For fish oil, there are no specific exclusions necessary except for those 
currently taking the supplement and unwilling to forego use during the trial.   
Potential side effects of the study agents will be assessed on each follow-up questionnaire. For 
fish oil, these side effects include gastrointestinal (GI) symptoms (stomach upset or pain, 
nausea, constipation, diarrhea), bleeding (any GI bleed, GI bleeding requiring transfusion, 
hematuria, easy bruising, epistaxis), skin rash, colds or upper respiratory tract infection, flu-like 
symptoms, bad taste in mouth, and physician diagnosis of atrial fibrillation or other irregular 
rhythms. For vitamin D, these side effects include GI symptoms as listed above, and physician 
diagnosis of hypercalcemia or kidney stones.  
There is also the possibility of social/psychological risk that could result from inadvertent 
disclosure of confidential information from the questionnaires or blood tests. However, we have 
many safeguards in place to avoid this possibility, and we have never had an inadvertent breach 
of confidentiality in any of our trials.  
With regard to the risks of the study agents, information on potential side effects discussed 
above will be elicited on each follow-up questionnaire for monitoring by the Data and Safety 
Monitoring Board.  We will compare the incidence of potential side effects in the active v. 
placebo groups for each agent, including the incidence of kidney stones with vitamin D 
assignment and the incidence of GI symptoms, skin abnormalities, and bleeding with fish oil 
assignment. 
The potential risk of disclosure of confidential information is guarded against by maintaining 
completed questionnaires and blood test results in locked offices  accessible by authorized 
personnel only. In questionnaire data files , participants are identified only by study ID. Subject 
identifiers are stored in separate computer files with password protection, and results will be 
presented only in the aggregate. In addition, employees involved with the proposed study will be 
asked to sign a form agreeing not to disclose any information to which they might have access, 
regardless of their personal perception about its confidentiality. Each employee of the study who 
has access to study data or has contact with subjects  participates in an institutional (Brigham 
and Women’s Hospital) human subjects educational program, which consists of reviewing 
regulatory and informational documents pertaining to human subjects research; passing a test 
on ethical principles and regulations governing human subjects research; and signing a 
statement of commitment to the protection of human subjects.  Finally, all such employees are 
required to participate in HIPAA training 
 
VIII. POTENTIAL BENEFITS 
 
For the majority of participants, there will be few direct benefits from participating in this primary 
prevention study other than the awareness of being involved in a large endeavor to answer 
relevant and timely questions regarding the possible benefit of vitamin D and fish oil. During the 
trial itself, we will receive inquiries from the participants regarding both specific and general 
health concerns. We will respond to each of those questions, primarily directing participants to 
published sources or recommending that they see their local health provider who is familiar with 
their medical history.  

 
18
The purported health benefits of vitamin D and marine omega-3 fatty acids are receiving 
increasing attention in the medical literature and the popular press. Sales of vitamin D 
supplements and omega-3 fatty acid supplements at U.S. stores have increased substantially in 
recent years. However, definitive data on health benefits and risks of these agents are lacking. 
Findings from this large clinical trial will clarify the role of vitamin D and marine omega-3 fatty 
acid supplements in the primary prevention of cancer and CVD in men and women and will help 
guide individual decisions, clinical recommendations, and public health guidelines. 
 
IX. MONITORING AND QUALITY ASSURANCE 
 
A. SAFETY MONITORING 
 
An independent Data and Safety Monitoring Board (DSMB) will be assembled, consisting of 
experts in clinical trials, epidemiology, biostatistics, relevant clinical areas of cancer and CVD, 
and NIH representatives. The Physicians’ Health Study, the Women’s Health Study, and the 
Women’s Antioxidant and Folic Acid Cardiovascular Study have been monitored by the same 
DSMB since their inception. As two of these trials have ended and the third is in its last years, 
we will ask our DSMB members if they would be willing to also monitor VITAL. If they are not 
able to do so, we will consult with them in assembling a new DSMB.  
The DSMB will annually examine the progress of the trial and the unblinded data on study 
endpoints and possible adverse effects to recommend continuation, alteration of study design, 
or early termination, as appropriate. Interim trial results will be assessed with the Haybittle-Peto 
rule,84, 85 adjusting for multiple looks. In this method, interim results are compared to a z-score of 
3 standard deviations (p=0.0027) throughout the trial. The final results may then be interpreted 
as having close to nominal significance levels. This rule appropriately requires very strong 
evidence for early stopping, is more conservative than the Pocock86 and O’Brien-Fleming87 rules 
and the alpha-spending function,88 and can be conducted at convenient times without inducing 
statistical complexity.89 
While these rules are intended for the primary endpoints, the goal of VITAL is to assess the 
overall balance of benefits and risks of the two agents in the primary prevention of cancer and 
CVD. Thus, consideration will also be given to the secondary endpoints that are needed in the 
interpretation of overall results. In addition, the monitoring rules will serve solely as guidelines in 
decisions regarding continuation or stopping of treatment arms. All decisions must be made 
after examining the totality of evidence, including other trial data, on these agents. 
 
B. MONITORING OF SOURCE DATA AND DRUG DISPENSEMENT 
 
Redundancies will be built into the data processing systems to insure accurate recording of data 
and proper follow-up. All research forms will be scanned in and the data read by a character-
recognition software program (Teleform). Out-of-range, internally inconsistent, and unclear data 
will be reviewed by a verifier who will edit misread variables. Forms that cannot be scanned, 
and name and address changes, will be entered using traditional double-entry procedures. All 
data will undergo additional within-form and across-time checks to verify accuracy. Following 
data entry, all questionnaire responses that require additional follow-up for missing data, 
participant comments on the form or for endpoint validation will be manually reviewed to insure 
correct processing and accurate follow-up. Address changes received from the post office or 
from participants will be manually keyed by data entry personnel and the resultant files 
compared and verified. 

 
19
 
Great care will be taken to insure that participants receive their randomly assigned drugs. Upon 
receipt of drug shipments from the manufacturer, each of the four drug groups will be stored in 
segregated areas. A random sample of drugs bottles or calendar packs will be pulled from each 
drug group and blindly tested to insure that the packaged contents match the study label. At the 
time of packaging for shipment to individual participants, listings will be divided according to 
drug group assignment and only one group will be packaged at a time. The packaging area will 
be cleaned and cleared prior to packaging and shipment of the next drug group. If drug 
packages are returned by the postal service as undeliverable or declined by a participant, the 
contents of the package will be blindly tested as an additional level of quality control. 
 
X. REFERENCES 
 
1. 
Davis CD, Hartmuller V, Freedman DM, Hartge P, Picciano MF, Swanson CA, Milner JA. 
Vitamin D and cancer: current dilemmas and future needs. Nutr Rev 2007; 65:S71-4. 
2. 
Institute of Medicine Food and Nutrition Board. Dietary Reference Intakes: Calcium, 
Phosphorous, Magnesium, Vitamin D and Fluoride. Washington DC: National 
Academies Press, 1999. 
3. 
Giovannucci E. Strengths and limitations of current epidemiologic studies: vitamin D as a 
modifier of colon and prostate cancer risk. Nutr Rev 2007; 65:S77-9. 
4. 
Looker AC, Dawson-Hughes B, Calvo MS, Gunter EW, Sahyoun NR. Serum 25-
hydroxyvitamin D status of adolescents and adults in two seasonal subpopulations from 
NHANES III. Bone 2002; 30:771-7. 
5. 
Holick MF. Vitamin D deficiency. N Engl J Med 2007; 357:266-81. 
6. 
Harris SS. Vitamin D and African Americans. J Nutr 2006; 136:1126-9. 
7. 
Moore CE, Murphy MM, Holick MF. Vitamin D intakes by children and adults in the 
United States differ among ethnic groups. J Nutr 2005; 135:2478-85. 
8. 
Martins D, Wolf M, Pan D, Zadshir A, Tareen N, Thadhani R, Felsenfeld A, Levine B, 
Mehrotra R, Norris K. Prevalence of cardiovascular risk factors and the serum levels of 
25-hydroxyvitamin D in the United States: data from the Third National Health and 
Nutrition Examination Survey. Arch Intern Med 2007; 167:1159-65. 
9. 
Harris SS, Dawson-Hughes B. Reduced sun exposure does not explain the inverse 
association of 25-hydroxyvitamin D with percent body fat in older adults. J Clin 
Endocrinol Metab 2007; 92:3155-7. 
10. 
Hedley AA, Ogden CL, Johnson CL, Carroll MD, Curtin LR, Flegal KM. Prevalence of 
overweight and obesity among US children, adolescents, and adults, 1999-2002. JAMA 
2004; 291:2847-50. 
11. 
Ogden CL, Carroll MD, Curtin LR, McDowell MA, Tabak CJ, Flegal KM. Prevalence of 
overweight and obesity in the United States, 1999-2004. JAMA 2006; 295:1549-55. 
12. 
Kris-Etherton PM, Harris WS, Appel LJ. Fish consumption, fish oil, omega-3 fatty acids, 
and cardiovascular disease. Circulation 2002; 106:2747-57. 
13. 
NIH Office of Dietary Supplements, National Heart Lung and Blood Institute. Working 
Group Report on Future Clinical Research Directions on Omega-3 Fatty Acids and 
Cardiovascular Disease. June 2, 2004. Bethesda MD. Available at: 
http://www.nhlbi.nih.gov/meetings/workshops/omega-3/omega-3-rpt.htm Accessed on 
March 28, 2008. 
14. 
Stipp D. Fish-oil doses can be hard to swallow. Wall Street Journal: January 8, 2008; 
Page D1-D2. 

 
20
15. 
Gosnell M. Top 100 science stories of 2007: #8: Can vitamin D save your life? Discover: 
December 12, 2007. Available at http://discovermagazine.com/2008/jan/year-in-science-
2007/can-vitamin-d-save-your-life Accessed March 25, 2008. 
16. 
Guthrie C. The 10 biggest medical breakthroughs. #10: Benefits of vitamin D. Time: 
December 24, 2007. Available at: 
http://www.time.com/time/specials/2007/top10/article/0,30583,1686204_1686252_16903
93,00.html  Accessed March 25, 2008. 
17. 
Harvard Health Letter. The top 10 health stories of 2006. Harv Health Lett 2006; 32:1-3. 
18. 
Frisinger C. New year, new you: 8 simple rules for a healthier 2008. #1. Get more 
vitamin D. Fort Worth Star-Telegram (Texas): January 1, 2008; Page E1. 
19. 
Brody JE. An oldie vies for nutrient of the decade. New York Times: February 19, 2008; 
Page F7. 
20. 
Stampfer MJ. Vitamin D in the spotlight. Newsweek: December 11, 2006. Available at: 
http://www.newsweek.com/id/43998 Accessed March 25, 2008. 
21. 
Peng T. Don't forget your vitamins. Newsweek: March 24, 2008; Page 78. 
22. 
Rosenfeld I. We may need more vitamin D. Parade Magazine: January 29, 2006; Page 
15. 
23. 
Rhodes P. Vitamin D essentials: what you need to know about this important nutrient 
and where best to obtain it. Cooking Light 2008; 22:44-50. 
24. 
Foreman J. Is there any way to tell whether you are getting enough vitamin D? Boston 
Globe: February 4, 2008; Page C2.  
25. 
Harvard Health Letter. A more D-manding diet. Some experts say we should have a lot 
more vitamin D in our diets because it's protective against several diseases. Harv Health 
Lett 2007; 32:3.  
26. 
Simopoulos AP, Robinson J. The Omega Diet: The Lifesaving Nutritional Program 
Based on the Diet of the Island of Crete. New York: HarperCollins, 1999. 
27. 
Sears B. The Omega Rx Zone: The Miracle of the New High-Dose Fish Oil. New York: 
HarperCollins, 2004. 
28. 
Kaufman M. Fish oil may aid against manic depression. Washington Post, 1999:Z07. 
29. 
Foreman J. Fatty acid imbalance hurts our health. Boston Globe: February 10, 2004. 
30. 
Bakalar N. Fish oil linked to lower Alzheimer's risk. New York Times: November 14, 
2006. 
31. 
Munoz SS. Shopping around: DHA-enhanced foods. Wall Street Journal: March 27, 
2008; Page D2. 
32. 
Canadian Cancer Society. Canadian Cancer Society Announces Vitamin D 
Recommendation. Available at: 
http://www.cancer.ca/ccs/internet/mediareleaselist/0,,3172_1613121606_1997621989_l
angId-en.html. Accessed May 6, 2008. 
33. 
Vieth R, Bischoff-Ferrari H, Boucher BJ, Dawson-Hughes B, Garland CF, Heaney RP, 
Holick MF, Hollis BW, Lamberg-Allardt C, McGrath JJ, Norman AW, Scragg R, Whiting 
SJ, Willett WC, Zittermann A. The urgent need to recommend an intake of vitamin D that 
is effective. Am J Clin Nutr 2007; 85:649-50. 
34. 
Davis CD, Dwyer JT. The "sunshine vitamin": benefits beyond bone? J Natl Cancer Inst 
2007; 99:1563-5. 
35. 
Bardia A, Tleyjeh IM, Cerhan JR, Sood AK, Limburg PJ, Erwin PJ, Montori VM. Efficacy 
of antioxidant supplementation in reducing primary cancer incidence and mortality: 
systematic review and meta-analysis. Mayo Clin Proc 2008; 83:23-34. 
36. 
Mosca L, Banka CL, Benjamin EJ, Berra K, Bushnell C, Dolor RJ, Ganiats TG, Gomes 
AS, Gornik HL, Gracia C, Gulati M, Haan CK, Judelson DR, Keenan N, Kelepouris E, 
Michos ED, Newby LK, Oparil S, Ouyang P, Oz MC, Petitti D, Pinn VW, Redberg RF, 
Scott R, Sherif K, Smith SC, Jr., Sopko G, Steinhorn RH, Stone NJ, Taubert KA, Todd 

 
21
BA, Urbina E, Wenger NK. Evidence-based guidelines for cardiovascular disease 
prevention in women: 2007 update. Circulation 2007; 115:1481-501. 
37. 
Kris-Etherton PM, Lichtenstein AH, Howard BV, Steinberg D, Witztum JL. Antioxidant 
vitamin supplements and cardiovascular disease. Circulation 2004; 110:637-41. 
38. 
Lee IM, Cook NR, Gaziano JM, Gordon D, Ridker PM, Manson JE, Hennekens CH, 
Buring JE. Vitamin E in the primary prevention of cardiovascular disease and cancer: the 
Women's Health Study: a randomized controlled trial. JAMA 2005; 294:56-65. 
39. 
Miller ER, 3rd, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E. Meta-
analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann 
Intern Med 2005; 142:37-46. 
40. 
Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C. Mortality in randomized 
trials of antioxidant supplements for primary and secondary prevention: systematic 
review and meta-analysis. JAMA 2007; 297:842-57. 
41. 
Yokoyama M, Origasa H, Matsuzaki M, Matsuzawa Y, Saito Y, Ishikawa Y, Oikawa S, 
Sasaki J, Hishida H, Itakura H, Kita T, Kitabatake A, Nakaya N, Sakata T, Shimada K, 
Shirato K. Effects of eicosapentaenoic acid on major coronary events in 
hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint 
analysis. Lancet 2007; 369:1090-8. 
42. 
Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico. Dietary 
supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial 
infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della 
Sopravvivenza nell'Infarto miocardico. Lancet 1999; 354:447-55. 
43. 
Lappe JM, Travers-Gustafson D, Davies KM, Recker RR, Heaney RP. Vitamin D and 
calcium supplementation reduces cancer risk: results of a randomized trial. Am J Clin 
Nutr 2007; 85:1586-91. 
44. 
Wactawski-Wende J, Kotchen JM, Anderson GL, Assaf AR, Brunner RL, O'Sullivan MJ, 
Margolis KL, Ockene JK, Phillips L, Pottern L, Prentice RL, Robbins J, Rohan TE, Sarto 
GE, Sharma S, Stefanick ML, Van Horn L, Wallace RB, Whitlock E, Bassford T, 
Beresford SA, Black HR, Bonds DE, Brzyski RG, Caan B, Chlebowski RT, Cochrane B, 
Garland C, Gass M, Hays J, Heiss G, Hendrix SL, Howard BV, Hsia J, Hubbell FA, 
Jackson RD, Johnson KC, Judd H, Kooperberg CL, Kuller LH, LaCroix AZ, Lane DS, 
Langer RD, Lasser NL, Lewis CE, Limacher MC, Manson JE. Calcium plus vitamin D 
supplementation and the risk of colorectal cancer. N Engl J Med 2006; 354:684-96. 
45. 
Hathcock JN, Shao A, Vieth R, Heaney R. Risk assessment for vitamin D. Am J Clin 
Nutr 2007; 85:6-18. 
46. 
Lang JM, Buring JE, Rosner B, Cook N, Hennekens CH. Estimating the effect of the run-
in on the power of the Physicians' Health Study. Stat Med 1991; 10:1585-93. 
47. 
Talwar SA, Aloia JF, Pollack S, Yeh JK. Dose response to vitamin D supplementation 
among postmenopausal African American women. Am J Clin Nutr 2007; 86:1657-62. 
48. 
Prentice RL. A case-cohort design for epidemiologic cohort studies and disease 
prevention trials. Biometrika 1986; 73:1-11. 
49. 
Rimm EB, Giovannucci EL, Stampfer MJ, Colditz GA, Litin LB, Willett WC. 
Reproducibility and validity of an expanded self-administered semiquantitative food 
frequency questionnaire among male health professionals. Am J Epidemiol 1992; 
135:1114-26. 
50. 
Feskanich D, Rimm EB, Giovannucci EL, Colditz GA, Stampfer MJ, Litin LB, Willett WC. 
Reproducibility and validity of food intake measurements from a semiquantitative food 
frequency questionnaire. J Am Diet Assoc 1993; 93:790-6. 
51. 
Salvini S, Hunter DJ, Sampson L, Stampfer MJ, Colditz GA, Rosner B, Willett WC. Food-
based validation of a dietary questionnaire: the effects of week-to-week variation in food 
consumption. Int J Epidemiol 1989; 18:858-67. 

 
22
52. 
Jacques PF, Sulsky SI, Sadowski JA, Phillips JC, Rush D, Willett WC. Comparison of 
micronutrient intake measured by a dietary questionnaire and biochemical indicators of 
micronutrient status. Am J Clin Nutr 1993; 57:182-9. 
53. 
Feskanich D, Willett WC, Colditz GA. Calcium, vitamin D, milk consumption, and hip 
fractures: a prospective study among postmenopausal women. Am J Clin Nutr 2003; 
77:504-11. 
54. 
Hunter DJ, Rimm EB, Sacks FM, Stampfer MJ, Colditz GA, Litin LB, Willett WC. 
Comparison of measures of fatty acid intake by subcutaneous fat aspirate, food 
frequency questionnaire, and diet records in a free-living population of US men. Am J 
Epidemiol 1992; 135:418-27. 
55. 
Vieth R. Critique of the considerations for establishing the tolerable upper intake level for 
vitamin D: critical need for revision upwards. J Nutr 2006; 136:1117-22. 
56. 
Bischoff-Ferrari HA, Giovannucci E, Willett WC, Dietrich T, Dawson-Hughes B. 
Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health 
outcomes. Am J Clin Nutr 2006; 84:18-28. 
57. 
Dawson-Hughes B, Heaney RP, Holick MF, Lips P, Meunier PJ, Vieth R. Estimates of 
optimal vitamin D status. Osteoporos Int 2005; 16:713-6. 
58. 
Giovannucci E, Liu Y, Rimm EB, Hollis BW, Fuchs CS, Stampfer MJ, Willett WC. 
Prospective study of predictors of vitamin D status and cancer incidence and mortality in 
men. J Natl Cancer Inst 2006; 98:451-9. 
59. 
Aloia JF, Patel M, DiMaano R, Li-Ng M, Talwar SA, Mikhail M, Pollack S, Yeh  
            JK. Vitamin D intake to attain a desired serum 25-hydroxyvitamin D concentration. Am J 
Clin Nutr 2008;87:1952-1958 
60. 
Harris WS. International recommendations for consumption of long-chain omega-3 fatty 
acids. J Cardiovasc Med (Hagerstown) 2007; 8 Suppl 1:S50-2. 
61. 
Lee JH, O'Keefe JH, Lavie CJ, Marchioli R, Harris WS. Omega-3 fatty acids for 
cardioprotection. Mayo Clin Proc 2008; 83:324-32. [Docket No. 86G-0289], 1997. 
62. 
Agency for Healthcare Research and Quality. Effects of Omega-3 Fatty Acids on 
Cardiovascular Disease. Evidence Report/Technology Assessment Number 94. AHRQ 
Pub. No. 04-E009-1. 2004. Available at: 
http://www.ahrq.gov/clinic/epcsums/o3cardsum.htm Accessed March 28, 2008. 
63. 
Simopoulos AP. Evolutionary aspects of diet, the omega-6/omega-3 ratio and genetic 
variation: nutritional implications for chronic diseases. Biomed Pharmacother 2006; 
60:502-7. 
64. 
Kris-Etherton PM, Taylor DS, Yu-Poth S, Huth P, Moriarty K, Fishell V, Hargrove RL, 
Zhao G, Etherton TD. Polyunsaturated fatty acids in the food chain in the United States. 
Am J Clin Nutr 2000; 71:179S-88S. 
65. 
Harris WS. The omega-6/omega-3 ratio and cardiovascular disease risk: uses and 
abuses. Curr Atheroscler Rep 2006; 8:453-9. 
66. 
Stanley JC, Elsom RL, Calder PC, Griffin BA, Harris WS, Jebb SA, Lovegrove JA, Moore 
CS, Riemersma RA, Sanders TA. UK Food Standards Agency Workshop Report: the 
effects of the dietary n-6:n-3 fatty acid ratio on cardiovascular health. Br J Nutr 2007; 
98:1305-10. 
67. 
Harris WS, Poston WC, Haddock CK. Tissue n-3 and n-6 fatty acids and risk for 
coronary heart disease events. Atherosclerosis 2007; 193:1-10. 
68. 
Mozaffarian D, Ascherio A, Hu FB, Stampfer MJ, Willett WC, Siscovick DS, Rimm EB. 
Interplay between different polyunsaturated fatty acids and risk of coronary heart 
disease in men. Circulation 2005; 111:157-64. 
69. 
Department of Health and Human Services, U.S. Food and Drug Administration. 
Substances affirmed as generally recognized as safe: menhaden oil. Federal Register. 
June 5, 1997. Vol 62, No 108: pp 30751-30757. 21 CFR Part 184 [Docket No. 86G-

 
23
0289], 1997.52. 
Jacques PF, Sulsky SI, Sadowski JA, Phillips JC, Rush D, Willett 
WC. Comparison of micronutrient intake measured by a dietary questionnaire and 
biochemical indicators of micronutrient status. Am J Clin Nutr 1993; 57:182-9. 
70. 
Harris WS. Expert opinion: omega-3 fatty acids and bleeding--cause for concern? Am J 
Cardiol 2007; 99:44C-46C. 
71. 
Bays HE. Safety considerations with omega-3 fatty acid therapy. Am J Cardiol 2007; 
99:35C-43C. 
72. 
Satterfield S, Greco PJ, Goldhaber SZ, Stampfer MJ, Swartz SL, Stein EA, Kaplan L, 
Hennekens CH. Biochemical markers of compliance in the Physicians' Health Study. Am 
J Prev Med 1990; 6:290-4. 
73. 
Fritz AG, Percy C, Jack A, Shanmugaratham K, Sobin LH, Parkin MD, Whelan S. 
International Classification of Diseases for Oncology (ICD-O), Third Edition. Geneva: 
World Health Organization, 2000. 
74. 
Thygesen K, Alpert JS, White HD, Joint ESC/ACCF/AHA/WHF Task Force for the 
Redefinition of Myocardial Infarction. Universal definition of myocardial infarction. 
Circulation 2007; 116:2634-53. 
75. 
Adams HP, Jr., Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, Marsh EE, 3rd. 
Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter 
clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke 1993; 24:35-
41. 
76. 
Cox DR. Regression models and life-tables. J Royal Statist Soc B 1972; 34:187-220. 
77. 
Gooley TA, Leisenring W, Crowley J, Storer BE. Estimation of failure probabilities in the 
presence of competing risks: new representations of old estimators. Stat Med 1999; 
18:695-706. 
78. 
Putter H, Fiocco M, Geskus RB. Tutorial in biostatistics: competing risks and multi-state 
models. Stat Med 2007; 26:2389-430. 
79. 
Fine JP, Gray RJ. A proportional hazards model for the subdistribution of a competing 
risk. J Am Stat Assoc 1999; 94:496-509. 
80. 
Barlow WE. Robust variance estimation for the case-cohort design. Biometrics 1994; 
50:1064-72. 
81. 
Borgan O, Langholz B, Samuelsen SO, Goldstein L, Pogoda J. Exposure stratified case-
cohort designs. Lifetime Data Anal 2000; 6:39-58. 
82. 
Holick MF. Vitamin D for health and in chronic kidney disease. Semin Dial 2005; 18:266-
75 
83. 
Freedman LS. Tables of the number of patients required in clinical trials using the 
logrank test. Stat Med 1982; 1:121-9. 
84. 
Haybittle JL. Repeated assessment of results in clinical trials of cancer treatment. Br J 
Radiol 1971; 44:793-7. 
85. 
Peto R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard SV, Mantel N, McPherson 
K, Peto J, Smith PG. Design and analysis of randomized clinical trials requiring 
prolonged observation of each patient. I. Introduction and design. Br J Cancer 1976; 
34:585-612. 
86. 
Geller NL, Pocock SJ. Interim analyses in randomized clinical trials: ramifications and 
guidelines for practitioners. Biometrics 1987; 43:213-23. 
87. 
O'Brien PC, Fleming TR. A multiple testing procedure for clinical trials. Biometrics 1979; 
35:549-56. 
88. 
DeMets DL, Lan KK. Interim analysis: the alpha spending function approach. Stat Med 
1994; 13:1341-52. 
89. 
Pocock SJ. Current controversies in data monitoring for clinical trials. Clin Trials 2006; 
3:513-21. 
 

 
1
 PROTOCOL FOR THE VITAL TRIAL  
November 30, 2017, version 23 
 
 
SUMMARY 
 
The VITAL trial (VITamin D and OmegA-3 TriaL) is a randomized, double-blind, placebo-
controlled, 2x2 factorial trial of vitamin D (in the form of vitamin D3 [cholecalciferol] softgel 
capsules) and marine omega-3 fatty acid (Omacor® eicosapentaenoic acid [EPA] + 
docosahexaenoic acid [DHA]) supplements in the primary prevention of cancer and 
cardiovascular disease (CVD). The trial will be conducted among 26,500 men aged ≥50 and 
women aged ≥55, to be recruited from mailings to 9.25 million persons, including health and 
other professionals, members of AARP (formerly known as the American Association of Retired 
Persons), and others. Willing and eligible respondents to the mailings will be enrolled in a 3-
month run-in, during which they will receive placebos. Upon successful completion of the run-in, 
participants who remain willing and eligible will be randomly assigned to one of four treatment 
groups for 5 years: vitamin D3 (2000 IU/d) and fish oil (1000 mg capsule containing Omacor® 
840 mg EPA+DHA); vitamin D3 and fish oil placebo; placebo vitamin D3 and fish oil; and placebo 
vitamin D3 and placebo fish oil. Blood samples will be collected and stored to allow assessment 
of effect modification by baseline 25-hydroxyvitamin D and omega-3 fatty acid levels, as well as 
future ancillary studies of genetic/biochemical hypotheses. At 1-year intervals, participants will 
receive a new supply of capsules and a follow-up questionnaire on compliance, possible side 
effects, and incidence of endpoints. Endpoints will be confirmed by medical record review.  
I. BACKGROUND AND SIGNIFICANCE 
The VITAL Trial will evaluate the role of vitamin D and long-chain marine omega-3 
polyunsaturated fatty acid (Omacor® eicosapentaenoic acid [EPA] plus docosahexaenoic acid 
[DHA]) supplements in the primary prevention of cancer and cardiovascular disease (CVD). 
Although there have been marked advances in our understanding of potentially beneficial 
effects of vitamin D and omega-3 fatty acids on cancer and cardiovascular risk in recent years, 
clear gaps in knowledge remain.  
Data from laboratory studies, epidemiologic investigations, and small clinical trials strongly 
suggest a protective effect for vitamin D against cancer, but there are no completed or ongoing 
large randomized trials of high-dose vitamin D supplements for the primary prevention of cancer 
in a general population.1 At a 2007 conference on the role of vitamin D in cancer prevention 
sponsored by the National Cancer Institute (NCI) and the National Institutes of Health Office of 
Dietary Supplements (ODS), most speakers “expressed a need for intervention studies with 
comprehensive assessment of vitamin D status.1 To quote one such investigator, Dr. Edward 
Giovannucci, “Further observational studies—and randomized trials if feasible—would be useful 
in testing the hypothesis that vitamin D lowers cancer risk. … Confirming that vitamin D reduces 
the risk of cancer incidence or mortality is critical, because current health recommendations 
typically do not encourage high intakes of vitamin D and tend to discourage sun exposure. 
Current dietary recommendations are geared only to prevent quite low vitamin D levels. If the 
association between better vitamin D status and reduced cancer risk is a causal one, the levels 
of intake currently recommended are probably inadequate. [Note: The 2011 Institute of Medicine 
guidelines call for 600 IU/d of vitamin D for adults aged 51-70 and 800 IU/d for adults aged >70 
to maintain bone health and normal calcium metabolism.2] Defining … the optimal levels of 
vitamin D for cancer prevention remains a challenge, but further study should be a high priority 
because the potential for benefit is substantial.”3 

 
2
Available data on the connection between vitamin D and CVD are less extensive but also 
support a possible protective effect. It is thus concerning that >1/2 of U.S. middle-aged and 
older women and >1/3 of similarly aged men have vitamin D insufficiency.4, 5  African-American 
(black) individuals are particularly vulnerable, because darkly pigmented skin is less able to 
synthesize vitamin D in response to solar radiation and because they tend to have lower intakes 
of dietary and supplemental vitamin D than whites.6, 7 Obese individuals are also at above-
average risk, presumably due to decreased bioavailability of this fat-soluble vitamin.8, 9 Given 
the aging population and soaring obesity prevalence,10, 11 low vitamin D status is an increasingly 
important public health issue. 
Marine omega-3 fatty acids have shown considerable promise for the primary prevention of 
CVD in laboratory and observational studies; large randomized trials in secondary prevention or 
high-risk settings also find benefit. However, there are no completed or ongoing trials of such 
supplements for the primary prevention of CVD in a general population that has been selected 
only on the basis of age and not on vascular risk factors such as diabetes. The Nutrition 
Committee of the American Heart Association (AHA) has stated that “Additional studies are 
needed to confirm and further define the health benefits of omega-3 fatty acid supplements for 
… primary … prevention.”12 (p. 2775) The conclusion from a 2004 workshop of the ODS and the 
National Heart, Lung and Blood Institute (NHLBI) Working Group on Future Clinical Research 
Directions on Omega-3 Fatty Acids and CVD13 is that “… the body of evidence is consistent with 
the hypothesis that intake of omega-3 fatty acids reduces CVD but … a definitive trial is needed” 
and that “A primary prevention trial [would] fill a major gap in knowledge because virtually none 
of the trials to date have been conducted in a primary prevention population and thus … it is 
unknown whether omega-3 fatty acids can prevent first events.” In terms of methodology, the 
ODS/NHLBI Working Group recommended a large simple trial, with fish oil given in supplement 
rather than in food form. It is critically important that such a trial be undertaken in a timely 
fashion, to avoid a possible dilution of effect from fortification of the food supply with omega-3 
fats, an increasingly common practice among U.S. manufacturers.14  
It is important to clarify the roles of vitamin D and omega-3 fatty acids in the primary prevention 
of cancer and CVD because their purported health benefits are receiving increasing attention in 
both the medical literature and the popular press.15-31 The Canadian Cancer Society recently 
raised its recommended dose of vitamin D to 1000 IU/d,32 and many experts are urging a similar 
move in the U.S.33 Sales of individual vitamin D supplements at U.S. grocery, drug, and “big 
box/club” stores jumped 91% to nearly $30 million during 2007 (C. Reider of Pharmavite LLC, 
personal communication). In 2007, sales of omega-3 fatty acid supplements in the U.S. reached 
$600 million, an increase of 20% from the prior year; omega-3 supplements are now the fifth-
best-selling dietary supplement.14 In addition, an increasing number of foods are omega-3 
fortified; >1200 such products were launched in 2006 alone.14 The growing enthusiasm for 
vitamin D and fish oil underscores the urgent need for a timely initiation of a large randomized 
trial to test these agents rigorously, before their use becomes so prevalent (through 
supplements and fortification of food) as to render participant recruitment and hypothesis testing 
impossible. As Dr. Cindy Davis of the NCI has noted, “While vitamin D may well have multiple 
benefits beyond bone, health professionals and the public should not in a rush to judgment 
assume that vitamin D is a magic bullet and consume high amounts of vitamin D. More definitive 
data on both benefits and potential adverse effects of high doses are urgently needed.”34 The 
same can be said of marine omega-3 fatty acids; the conclusion from a 2004 workshop on 
omega-3 fatty acids and CVD sponsored by ODS and the NHLBI was that “a definitive trial is 
needed.”13  

 
3
Indeed, skeptics will note that most single-agent nutritional interventions have not withstood the 
test of rigorous trials for the primary prevention of cancer and CVD—witness the disappointing 
findings for antioxidant vitamins.35-40 However, unlike the antioxidant scenario, the two agents in 
this proposal appear promising not only in observational studies but also in randomized settings. 
Omega-3 fatty acids have shown cardiovascular benefit in two large trials in high-risk 
individuals41, 42 (one of which also tested vitamin E and found no benefit for it42), while vitamin D 
was suggestive of cancer benefit in a modestly sized trial43 and of reductions in total and cancer 
mortality even at a low dose in another.44 The proposed large-scale randomized controlled trial 
of vitamin D and omega-3 fatty acids will provide definitive data on their roles in the primary 
prevention of cancer and CVD. 
II. SPECIFIC AIMS 
 
The overall aim of the VITAL trial is to evaluate the role of vitamin D softgel capsules and long-
chain marine omega-3 polyunsaturated fatty acid (Omacor® eicosapentaenoic acid [EPA] plus 
docosahexaenoic acid [DHA]) supplements in the primary prevention of cancer and 
cardiovascular disease (CVD). 
Primary aims: 
1. To test whether vitamin D3 supplementation reduces the risk of (a) total cancer 
(excluding non-melanoma skin cancer) and (b) major CVD events (a composite endpoint 
of myocardial infarction [MI], stroke, and cardiovascular mortality). 
2. To test whether EPA+DHA supplementation reduces the risk of (a) total cancer 
(excluding non-melanoma skin cancer) and (b) major CVD events. 
Secondary aims: 
1. To test whether vitamin D3 or EPA+DHA supplementation reduces the risk of (a) incident 
colorectal cancer, (b) incident breast cancer (in women), (c) incident prostate cancer (in 
men), and (d) total cancer mortality. 
2. To test whether vitamin D3 or EPA+DHA supplementation reduces the risk of (a) an 
expanded composite cardiovascular endpoint of MI, stroke, cardiovascular mortality, and 
coronary revascularization (i.e., coronary artery bypass grafting [CABG] or percutaneous 
coronary intervention [PCI]) and (b) the individual components of the primary 
cardiovascular endpoint, particularly total CVD mortality. 
Tertiary aims: 
1. To explore whether vitamin D3 and EPA+DHA supplementation exhibit synergistic or 
additive effects on the risk of (a) total cancer, (b) major CVD events, and (c) the 
secondary endpoints specified above. 
2. To explore whether the effect of vitamin D3 or EPA+DHA supplementation on cancer and 
CVD risk varies by (a) baseline blood levels of these nutrients, (b) race/skin 
pigmentation (for vitamin D3), and (c) body mass index (BMI) (for vitamin D3). 
3. To explore whether vitamin D3 and EPA+DHA supplementation reduces the risks of 
heart failure. 
 
III. SUBJECT SELECTION 
 

 
4
Study population: VITAL will be conducted among 26,500 apparently healthy participants. 
Included in the trial will be men aged ≥50 and women aged ≥55, ages at which rates of chronic 
disease increase substantially. Excluded from the study will be individuals who have (1) a 
history of cancer (except non-melanoma skin cancer), MI, stroke, TIA, CABG, or PCI; (2) have 
any of the following safety exclusions: use of anticoagulants at baseline, history of renal failure 
or dialysis, hypercalcemia, hypo- or hyperparathyroidism, severe liver disease (cirrhosis), or 
sarcoidosis or other granulomatous diseases such as active chronic tuberculosis or Wegener’s 
granulomatosis; (3) have an allergy to fish (for EPA+DHA); (4) have another serious illness that 
would preclude participation; (5) are consuming greater than 800 IU of vitamin D from all 
supplemental sources combined (individual vitamin D supplements, vitamin D analogues, 
calcium+vitamin D supplements, medications with vitamin D [e.g., Fosamax Plus D], and 
multivitamins), and are not willing to decrease or forego such use during the trial (to ensure total 
supplemental vitamin D intake does not exceed the current safety limit [i.e., tolerable upper 
intake level of 4000 IU/d set by the Institute of Medicine2); (6) are consuming greater than 1200 
mg/d of calcium (the RDA for men aged >70 years and women aged >50 years2) from all 
supplemental sources combined; (7) are taking fish oil supplements and are not willing to forego 
their use during the trial; and (8) are not willing to participate, as evidenced by not signing the 
informed consent form. Although thiazide diuretics and supplemental vitamin D may interact to 
increase hypercalcemia risk, it is unlikely that such an interaction would occur at the vitamin D 
dose we propose.45 Thus, thiazide use will not be an exclusion criterion. 
 
Cohort assembly: A master mailing tape containing 9.25 million names and addresses will be 
assembled by Listmart, a list broker with access to all commercially available U.S. mailing lists. 
The listings will include licensed health professionals (e.g., physicians, nurses, pharmacists, 
veterinarians, physical therapists), other professionals (e.g., executives, managers, financial 
service professionals, computer professionals, architects, engineers, accountants), AARP 
members, and subscribers to magazines that have minority readership or appeal to 
professionals and college-educated individuals (e.g., Essence, National Geographic and Time). 
Additionally, it will include individuals who have indicated interest in participating through the 
study website (www.vitaltrial.org; see paragraph below), publicized via advertisements and 
feature articles in “healthy-lifestyle” publications, as well as individuals who are currently 
participating in our other observational epidemiologic studies (e.g., Women’s Health Study). Of 
the 9.25 million names on the mailing tape, an estimated 30% or more will be black or Hispanic. 
Each invited individual will be assigned a study identification number and mailed an invitation 
letter and screening questionnaire. 
 
In addition, we will place advertisements and feature articles in magazines that have minority 
readership and in “healthy-lifestyle” publications, as well as setting up a booth at health fairs and 
distributing fliers on the study. The advertisement/flier would explain the objectives of the trial 
and the general eligibility criteria. Interested individuals would be encouraged to visit a study 
web site at www.vitaltrial.org where they could review in greater detail the purpose of the trial, 
what participation involves, and the eligibility criteria. By submitting their name, address and age 
at the web site, individuals would be assigned a study identification number and mailed the 
VITAL invitation letter and a screening questionnaire. If necessary, we will also consider inviting 
eligible participants from groups with large minority representation, such as church groups. 
 
IV. SUBJECT ENROLLMENT 
 
Screening : The W.A. Wilde Company, a direct marketer in Holliston, MA that we have used in 
our previous trials, will mail screening  materials by non-profit bulk rate to each individual in the 

 
5
assembled cohort. The mailings will occur in groupings of approximately ≥300,000 each, spaced 
at monthly intervals. The mailing package will include: 
• 
A letter explaining the rationale for VITAL. The letter will outline what participation would 
entail. We will also send a targeted letter to individuals identified as African-American 
with a high degree of certainty (e.g., Essence subscribers). The special invitation letter 
would emphasize that African-Americans are at higher risk for vitamin D deficiency, as 
well as heart disease, stroke, and certain cancers.  
• 
A very brief screening questionnaire with items on demographics (age, gender, 
race/ethnicity, education); medical history (cancer, MI, stroke, CABG and PCI); and 
current use of supplements containing vitamin D or fish oil.  
• 
A self-addressed, pre-paid envelope for returning study forms. 
If the initial response rates are lower than anticipated, a second enrollment request will be 
mailed to selected non-respondents, who will be identified from mailing groups that had high 
response and eligibility rates. 
Eligible at screening : A computer program will evaluate the screening questionnaire data to 
classify respondents’ eligibility to receive the baseline questionnaire and informed consent form 
as ineligible, eligibility pending, or eligible. Respondents with eligibility pending will be sent a 
follow-up form or called to clarify incomplete or inconsistent responses so that they can be 
classified as ineligible or eligible. Respondents will be eligible to receive the baseline 
questionnaire and informed consent form if they meet these criteria: (1) are age ≥50 (men) or 
≥55 (women) years; (2) have no history of cancer (except non-melanoma skin cancer), MI, 
stroke, CABG, or PCI; (3) are consuming no more than 800 IU of vitamin D from all 
supplemental sources combined (individual vitamin D supplements, vitamin D analogues, 
calcium+vitamin D supplements, medications with vitamin D [e.g., Fosamax Plus D], and 
multivitamins), or, if taking, willing to decrease or forego such use during the trial (to ensure total 
supplemental vitamin D intake does not exceed the current safety limit [i.e., tolerable upper 
intake level of 4000 IU/d set by the Institute of Medicine2); (4) are not taking fish oil 
supplements, or, if taking, willing to forego their use during the trial.  
We estimate that approximately 160,000 respondents will be potentially eligible  for participation 
in the VITAL trial based upon their responses on the screening questionnaire. Respondents who 
are not eligible will be mailed a letter thanking them for their interest and informing them of the 
general criteria that determined eligibility. 
Enrollment: At periodic intervals, the names/addresses of recent respondents, who are eligible 
to receive the baseline questionnaire, will be electronically compiled in preparation for the 
mailing of the baseline questionnaire and informed consent form. It is anticipated that there will 
be 5-10,000 entries per bi-weekly list. W.A. Wilde Company will mail these materials by non-
profit bulk rate to each: 
• 
A letter of appreciation for the initial response which also reiterates the purpose of the 
study and what participation involves.  
• 
An informed consent form. 
• 
A baseline questionnaire with items on demographics (date of birth); medical history 
(kidney stones, hypercalcemia, kidney failure, sarcoidosis, liver disease, thyroid disease, 
chronic tuberculosis, other major illnesses); allergy to fish; current use of calcium 
supplements and other supplements containing vitamin D or fish oil; height and weight; 

 
6
use of anticoagulant medications; and potential effect modifiers such as skin 
pigmentation and sunlight exposure. Respondents will also be asked for telephone 
number(s) and an e-mail address in the event that follow-up needs to be conducted in 
order to clarify questionnaire responses and to provide their social security number 
(optional) which will be used for identification purposes only. In addition, the form will 
contain a question asking whether individuals would be willing to return a blood sample if 
mailed a convenient blood collection kit. 
• 
A self-addressed, pre-paid envelope for returning study forms. 
Eligibility: A computer program will evaluate the questionnaire data to classify respondents’ 
eligibility as ineligible (to enter the run-in), eligibility pending, or eligible. The “eligibility pending” 
respondents will be sent a follow-up form to clarify incomplete or inconsistent responses. 
Respondents will be eligible if they meet these criteria: (1) have none of the following safety 
exclusions: use of anticoagulants, history of renal failure or dialysis, hypercalcemia, hypo- or 
hyperparathyroidism, severe liver disease (cirrhosis), or sarcoidosis or other granulomatous 
diseases such as active chronic tuberculosis or Wegener’s granulomatosis; (2) have no allergy 
to fish (for EPA+DHA) or fish oil supplements; (3) have no other serious illness that would 
preclude participation; (4) are consuming no more than 800 IU of vitamin D from all 
supplemental sources combined (individual vitamin D supplements, vitamin D analogues, 
calcium+vitamin D supplements, medications with vitamin D [e.g., Fosamax Plus D], and 
multivitamins), or, if taking, willing to decrease or forego such use during the trial (to ensure total 
supplemental vitamin D intake does not exceed the current safety limit [i.e., tolerable upper 
intake level of 4000 IU/d set by the Institute of Medicine2); (5) are consuming no more than 1200 
mg/d of calcium (the RDA for men aged >70 years and women aged >50 years2) from all 
supplemental sources combined; (6) are willing to participate, as evidenced by signing the 
informed consent form.  
We estimate that approximately 40,000 individuals will be both willing and fully eligible to 
participate in the trial and enter the run-in. Respondents who are not eligible will be mailed a 
letter thanking them for their interest and informing them of the general criteria that determined 
eligibility. 
Run-in: The experience of other studies conducted by our research group (PHS, WHS, and 
WAFACS) has demonstrated the success of a run-in period in identifying a group of excellent 
compliers for long-term follow-up. Because the health experience of participants will be 
analyzed according to randomized treatment assignment, the use of a run-in phase to eliminate 
poor compliers prior to randomization will increase the power of the study.46 During a 3-month 
run-in period, all participants will take placebo vitamin D softgel capsules and placebo fish oil. 
As the effects of both agents on cancer prevention are likely to be chronic, it would not be 
scientifically appropriate to use active agent in the run-in and then randomize to placebo. 
Moreover, the use of placebos will allow us to most clearly analyze the side effects of the study 
agents during the randomized treatment period.  
At trial enrollment, willing and eligible participants will be mailed a 6-month supply of run-in 
calendar packs (i.e., 3 months for the actual run-in and 3 months to allow pill-taking to continue 
uninterrupted while we check participants’ responses for continued eligibility and then ship 
randomized capsules to participants who remain eligible). Participants will take two capsules per 
day (one vitamin D placebo softgel capsule and one fish oil placebo capsule). At 3 months, 
participants will receive a follow-up run-in questionnaire asking about continued eligibility, 

 
7
willingness to continue in the trial, pill compliance, and potential side effects. Non-responders 
will be mailed up to 3 requests at 6-week intervals.  
Participants will be randomized only if they: (1) demonstrate good compliance in pill taking, 
defined as taking at least two-thirds of the study pills during the run-in (i.e., missed 10 or fewer 
days of pill taking in the past month); (2) express willingness to continue in the trial; (3) report no 
new history of cancer (except non-melanoma skin cancer), MI, stroke, TIA, CABG, PCI, 
hypercalcemia, sarcoidosis, kidney failure or dialysis, parathyroid disease, cirrhosis or other 
severe liver disease, or other serious illness, use of anticoagulants or vitamin D analogues,  
during the run-in; (4) are consuming no more than 1200 mg/d of calcium (the RDA for men aged 
>70 years and women aged >50 years2) from all supplemental sources combined; and (5) 
demonstrate continued willingness to limit supplemental vitamin D intake to ≤800 IU/d and to 
forego the use of fish oil supplements. 
Because we are close to reaching our original target sample of 20,000 (now revised to 26,500) 
participants randomized, but have not yet reached our target of 25% African-American 
participants, we will modify the compliance criterion to be more strict for white, non-Hispanic 
participants (starting October 2012) as follows: White, non-Hispanic participants who have not 
provided a blood sample will be randomized only if they demonstrate good compliance in pill 
taking, defined as taking at least 83% of study pills during the run-in (i.e., missed 5 or fewer 
days of pill taking in the past month). For white, non-Hispanic participants living in the 
catchment area for clinic visits (approved under protocol 2010P002331), the original compliance 
criterion (taking at least two-thirds of the study pills during the run-in pills) will still apply if they 
are willing to participate in a clinic visit. 
We estimate that of the 40,000 initially willing and eligible individuals enrolled in the run-in, 66% 
(n=26,500) will be compliant and remain willing and eligible for randomization.  
Randomization: Willing and eligible participants will be randomly assigned to one of four 
treatment groups: vitamin D3 softgel capsules (2000 IU/d) and fish oil (Omacor® EPA+DHA, 840  
mg/d); vitamin D3 softgel capsules and fish oil placebo; placebo vitamin D3 softgel capsules and 
Omacor® fish oil; and placebo vitamin D3 softgel capsules and placebo fish oil. Individuals will 
be randomized to treatment within 5-year age groups using a computer-generated table of 
random numbers. Within each age group, treatment assignments will be generated in blocks of 
eight individuals, with two individuals in each of the four treatment combinations. The date of 
randomized treatment assignment will be the formal “in-study” date. (The use of age 
stratification during randomization will increase statistical efficiency and power.) Participants will 
be mailed an annual supply of capsules (in calendar packs) and instructed to take two capsules 
– one vitamin D softgel capsule or placebo and one of Omacor® fish oil or placebo – each day.  
V. STUDY PROCEDURES 
A. BLOOD COLLECTION  
Baseline: Participants who indicate on their baseline questionnaire a willingness to provide an 
optional venous blood sample will be sent a blood collection kit during the run-in. Participants 
who indicate on the questionnaire that they are “not sure” about participating or unwilling to 
participate in the blood collection will be mailed a letter informing them that we can arrange to 
have their blood drawn at home by a technician from a national medical service company, if 
they are now willing. The blood collection kit will include a consent form, supplies and 

 
8
instructions for having the blood drawn into two EDTA tubes, one heparin tube, and one serum 
tube, a gel-filled freezer pack, and an overnight courier air bill. We anticipate that most 
participants will have their blood drawn by their own healthcare providers or in another 
healthcare or blood-drawing facility. For those not able to do this on their own, VITAL staff will 
contact participants to schedule a visit to a blood-drawing facility, or to schedule a service for 
drawing blood from the participant at home. Participants will be instructed to provide a fasting 
sample, if possible, and to record the time of venipuncture and the time of their last meal. They 
will be instructed to send their sample to our blood laboratory via overnight courier in the freezer 
packs on the same day as the blood draw. Upon receipt, the samples will be centrifuged to 
separate plasma, red blood cells, and buffy coat; aliquoted into 2-ml Nunc vials (including 
multiple vials of EDTA heparin and serum plasma vials, and vials for EDTA red blood cells and  
buffy coat, and stored in individual vial locations in nitrogen freezers(-170 ºC). The process will 
be completed within several hours of specimen receipt to ensure that samples are frozen within 
30-36 hours after venipuncture. Each freezer has an electronic alarm which is monitored 24 
hours a day. If problems arise, staff members are paged by the alarm company and can adjust 
the levels of liquid nitrogen as needed. This system has protected stored samples from 
inadvertent thawing or warming for nearly 20 years.  
The main reason for collecting pre-randomization blood specimens is to provide the opportunity 
to assess whether treatment effects are modified by baseline blood levels of 25(OH)D and 
EPA+DHA. When stratifying by 25(OH)D, season (assessed by date of return) and geographic 
location (assessed by zip code) will be considered. This resource will also be extremely 
valuable for future applications for funding of ancillary studies to explore other genetic and 
biochemical hypotheses in this well-characterized cohort. A nested case-control or case-cohort 
approach can be used, enabling the study of many promising biochemical and genetic markers 
(e.g., VDR polymorphisms) as potential predictors of disease risk and modifiers of intervention 
effects at very low incremental cost. 
Follow-up: An optional follow-up blood sample will be collected in year 2 and year 4  from a  
subset of 6000 participants who provided a baseline fasting blood sample. These follow-up 
samples, to be collected using the same methods as for the baseline samples, will allow 
assessment of (a) pill-taking compliance, (b) changes in biomarkers with treatment, and (c) in 
the placebo group, the effect of changing trends in background fortification with vitamin D and 
omega-3 fats. The samples will be particularly important for an exploratory analysis of 
treatment-induced changes in 25(OH)D levels among black participants, as there are few data 
on this topic.47 In addition, changes in blood calcium and parathyroid hormone (PTH) levels will 
be examined as markers of possible hypercalcemia, a potential side effect of high vitamin D 
intake. The bloods will also be archived for future ancillary studies to evaluate whether 
treatment with one or both of the study agents leads to changes in biochemical factors, 
including those in the lipid, glucose tolerance, inflammation, endothelial dysfunction, thrombosis, 
insulin, and insulin-like growth factor pathways. 
Biochemical assays: Blood levels of 25(OH)D and EPA+DHA will be assayed on participants 
who provide an analyzable sample at baseline and all participants who provide a blood sample 
at follow-up. In addition, baseline and follow-up blood levels of calcium, PTH and lipoproteins 
will be assayed. These assays will be performed by Quest Diagnostics. Quest Diagnostics will 
also perform albumin, creatinine, liver function and lipid tests on a subset of participants. In 
addition, we will measure several biomarkers related to CVD (including cholesterol lipoprotein 
subfractions using the Vertical Auto Profile cholesterol test, apolipoproteins, thyroid function, 
vitamin D, albumin, creatinine, PCSK9 and a basic metabolic panel). These additional 

 
9
measurements will be done at Atherotec Diagnostics Lab in Birmingham, AL, which will fund the 
costs of these studies. In addition, PCSK (plasma-based genotype will be assayed on 
participants who additionally gave genetic consent. 
 
We will also measure metabolomic and lipidomic biomarkers on baseline and follow-up stored 
samples. Samples will be sent in a de-identified manner and measurements will be done at Dr. 
Mohit Jain’s laboratory at the University of California San Diego, and at LabCorp, Inc., Raleigh, 
NC. In a small subset of participants, we will also 1) measure cholesterol efflux and epigenetic 
biomarkers on participants who gave genetic consent by sending de-identified samples to our 
academic collaborators in Quebec, Dr. Benoit Arsenault and Dr. Luigi Bouchard (Institut 
universitaire de cardiologie et de pneumologie de Québec); and 2) measure additional vitamin D 
biomarkers on de-identified samples sent to our academic collaborators at Massachusetts 
General Hospital, Dr. Masao Kaneki and Dr. Sadeq Quraishi. 
Over 5 years of follow-up, we would expect to accrue approximately 1660 cancer cases and 990 
CVD cases, with somewhat smaller numbers if the agents are highly effective. If 60% of 
participants provide blood specimens, this would include about 996 cancer cases and 594 CVD 
cases with bloods.  
B. ASSESSMENT OF DIETARY AND SUPPLEMENTAL INTAKE 
 
A modified version of a validated, self-administered semi-quantitative food frequency 
questionnaire (FFQ) will be mailed to participants during the run-in. The original questionnaire, 
developed by Walter Willett, MD, DrPH, and colleagues, is an efficient, reliable, and accurate 
instrument for categorizing individuals according to their intake of 32 nutrients, including vitamin 
D and marine omega-3 fats.48-53 Participants will be asked to estimate their average intake over 
the past year of various foods, beverages, and supplements that contain vitamin D, marine 
omega-3 fats, and other nutrients. Dietary vitamin D intake will be estimated from participants’ 
reported intakes of certain foods, including dairy products, fortified breakfast cereals, fortified 
juices, dark-meat fish, and cod liver oil. Dietary marine omega-3 fat intake will be estimated from 
intake of dark-meat fish, canned tuna, other fish, and seafood. Classifying participants by 
baseline intake of various nutrients will allow us to evaluate whether the study agents’ effects 
vary by such intake. Participants will also be asked to complete the FFQ at 4+ years, enabling 
an examination of dietary changes over time. 
Questions on use of non-study supplements or drugs containing vitamin D or EPA+DHA will be 
asked at baseline, 6 months, and on yearly follow-up questionnaires. We will also assess 
flaxseed and alpha-linolenic acid intakes. We will ascertain and analyze intake from food and 
supplements separately, and from both sources combined. 
 
C. TREATMENT 
In our research application, we proposed testing 1600 IU (40 μg) per day of vitamin D3 
(cholecalciferol) in VITAL. Since the application was submitted, based on new research and with 
approval from NCI, we now will test 2000 IU (50 µg) per day instead. We seek to obtain a large-
enough difference in vitamin D status between the treatment and placebo groups to detect 
health benefits, and there appear to be few safety issues associated with this dose. Recent 
studies have found little toxicity risk with doses up to 10,000 IU/d.45 (Nevertheless, to further 

 
10
ensure participants’ safety, we will exclude from the trial persons with a history of 
hypercalcemia, or sarcoidosis ; follow-up calcium and parathyroid hormone [PTH] levels will be 
obtained in a random subsample of participants.) Accumulating evidence suggests that vitamin 
D intakes well above currently recommended amounts of 600 IU/d for adults aged 51-70 and 
800 IU/d for adults aged >702 are necessary for health benefits. In a review of studies of serum 
25(OH)D in relation to bone mineral density, lower-extremity function, dental health, and risk of 
falls, fractures, and colorectal cancer, Bischoff-Ferrari et al.54 found that, for all endpoints, 
advantageous 25(OH)D levels began at 75 nmol/L, and optimal levels were between 90-100 
nmol/L. The average older individual requires an oral vitamin D3 intake of at least 800-1000 IU/d 
(20-25 µg) to achieve a serum 25(OH)D of 75 nmol/L.55 Among postmenopausal women in the 
WHI, 400 IU/d of vitamin D3 raised median plasma 25(OH)D from 42.3 to only 54.1 nmol/L.44, 56 
The primary reason for the dose change is a recent study by Aloia et al57 which indicates that 
the dose-response for serum 25(OH)D with vitamin D intake is not linear, but that the rate of 
increase in serum levels decreases at higher levels of intake. At a dose of 800 IU (20 μg/day), 
the rate of change is 1.2 nmol/L.  However, at doses ≥1,400 IU (35 μg/day), the rate of change 
decreases to 0.7 nmol/L.  Thus, extrapolation of these data suggests that with the originally 
proposed 1600 IU/d (40 μg/day), the expected change in mean serum 25(OH)D levels would be 
only 28 nmol/L, or a change from an estimated 42 nmol/L at baseline to a final mean level of 70 
nmol/L.  With 2000 IU/d (50 μg/day), the increase would be 35 nmol/L from baseline to a final 
mean level 77 nmol/L, which would be above the 75 nmol/L threshold level at which health 
benefits are believed to occur 54 , as well as a level believed to be free of any risks. 
 
Potential side effects of vitamin D are rare.  They include gastrointestinal (GI) upset (presence 
or absence of symptoms of peptic ulcer, nausea, constipation, diarrhea); and physician 
diagnosis of hypercalcemia or kidney stones, both of which are not expected to occur at the 
dose used in VITAL.2 To minimize the risks of hypercalcemia and kidney stones, we will require 
that participants limit their total intake of calcium from all sources, including multivitamins, single 
supplements of calcium, and other drugs that contain calcium to 1200 mg or less a day. 
Active vitamin D3 (2000 IU [50 µg] per softgel capsule) and matching inert placebo will be 
provided by Pharmavite LLC.  
 
In our research application, we proposed testing a total EPA+DHA dose of 1 mg/d (EPA [500 
mg/d] + DHA [500 mg/d]). Since the application was submitted, we have worked with various 
drug companies to develop soft-gel capsules containing these doses. We now propose to test a 
total dose of Omacor® EPA+DHA, 840 mg/d instead, since this is the highest dose of EPA + 
DHA currently available in a 1000 mg capsule. 
We believe that this dose provides the best balance of feasibility, efficacy and safety. We seek 
to obtain a large-enough difference in omega-3 fatty acid status between the treatment and 
placebo groups to detect health benefits, and there appear to be few safety issues associated 
with this dose. Health authorities recommend 400 mg to 1 g/d for cardioprotection.58 A total 
dose of 850 mg/d was used in the GISSI-Prevenzione Trial (EPA to DHA ratio, 1:2) and AREDS 
2 (EPA to DHA ratio, 2:1), while a dose of 1.8 g/d of EPA was used in JELIS. For this trial, we 
have selected a total dose of omega-3 fatty acids recommended by the American Heart 
Association for cardioprotection and demonstrated to be beneficial in one secondary prevention 
population with minimal side effects.42 Because the optimal ratio of EPA to DHA is unknown,59,60 
we have selected a 40:30 ratio of EPA to DHA. The ratio of omega-3 to omega-6 fatty acid 
intake is between 1:10 and 1:20 in most Western countries, including the U.S., whereas the 

 
11
optimal ratio for disease prevention has been hypothesized to be closer to 1:1 or 1:2,61,62 
although this is controversial.63 Indeed, there is growing consensus that the absolute intake of 
omega-3 is a more important predictor of health than is the ratio of omega-3 to omega-6 intake, 
at least for cardiovascular outcomes.63-66 However, given that the average intake of EPA+DHA 
is 100-200 mg/d among U.S. adults,58 the proposed intervention of 840 mg/d would be expected 
to increase the average participant’s omega-3 intake by a factor of 4 to 8. Assuming no 
concurrent change in omega-6 intake, the proposed omega-3 dose would thus have the effect 
of achieving the purported optimal omega-3 to omega-6 ratio and providing intakes associated 
with benefits in previous studies. 
As with vitamin D, potential side effects of omega-3 fatty acids are rare.  They include 
gastrointestinal (GI) symptoms (burping, stomach upset or pain, nausea, constipation, diarrhea), 
bleeding (any GI bleed, GI bleeding requiring transfusion, hematuria, easy bruising, epistaxis), 
skin rash, colds or upper respiratory tract infection, flu-like symptoms, and bad taste in mouth.  
The FDA has concluded that marine omega-3 fatty acid doses of up to 3 g/d are “Generally 
Recognized as Safe.”67  The AHA also has concluded that these risks are very low or low at 
doses of up to 1 g/d and low to moderate at doses of 1-3 g/d.12  Because they undergo an 
extensive purification process, high-quality fish oil supplements are free of environmental toxins 
(e.g., methylmercury, polychlorinated biphenyls [PCBs], and dioxins) found in some fish.12, 68  
Active fish oil (1000 mg capsule containing Omacor® 840 mg EPA + DHA ) and matching inert 
placebo will be provided by Pronova BioPharma ASA of Norway.  
 
D. FOLLOW-UP 
With regard to follow-up, at 6 months, participants will be mailed a very brief follow-up 
questionnaire and at each anniversary date a more detailed questionnaire and a re-supply of 
calendar packs. The questionnaire will include items on compliance with randomized 
treatments, use of non-trial supplements of vitamin D and fish oil, use of calcium supplements, 
development of major illnesses, dietary intakes of vitamin D and fish, and updated risk factors 
related to cancer and CVD. Information on potential side effects of the study agents will be 
elicited for monitoring by the Data and Safety Monitoring Board. For fish oil, these side effects 
include GI symptoms (burping, stomach upset or pain, nausea, constipation, diarrhea), bleeding 
(any GI bleed, GI bleeding, hematuria, easy bruising, epistaxis), skin rash, colds or upper 
respiratory tract infection, flu-like symptoms, and bad taste in mouth. For vitamin D, these side 
effects include GI symptoms, and physician diagnosis of hypercalcemia or kidney stones, both 
of which are not expected to occur at the dose used in VITAL.2 Non-responders will be mailed a 
second and third request and will then be telephoned to collect study data. If phone contact 
fails, a fourth request will be mailed in order to ascertain the participant’s study status and to 
obtain minimum information regarding the primary study outcomes. Participants will also be 
given the option of completing an electronic questionnaire sent via e-mail. These data will be 
collected and managed using REDCap electronic data capture tools. REDCap (Research 
Electronic Data Capture) is a secure, HIPAA-compliant web-based application hosted by 
PartnersHealthCare Research Computing.   
Internet sources will be used to assist in the tracking (new address, new phone number) of 
participants who are potentially lost to follow-up. At the very least, vital status will be 
ascertained. Participants will be instructed to discontinue their study capsules if, during follow-
up, they receive a diagnosis of hypercalcemia, sarcoidosis, or other safety-exclusion conditions 
and to inform their physicians about their participation in VITAL. If, in the course of follow-up, 

 
12
participants report taking supplementary vitamin D (not from diet) that amounts to greater than 
800 IU per day or daily calcium supplements that amount to greater than 1200 mg per day, they 
will be asked to limit their intakes to study guideline levels. These instructions will be included in 
the study guidelines which accompany each shipment of calendar packs. 
At 6-month intervals between annual follow-ups, participants will be mailed an interim form with 
questions on the development of primary endpoints (cancer, MI, and stroke), difficulties with pill 
compliance, and address changes. Participants will be asked to return the form only if one or 
more questions apply. The interim follow-up will help the staff maintain accurate contact 
information and provide a head start in addressing compliance issues and timely collection of 
medical records to confirm endpoints.  
A newsletter will be mailed to participants twice each year. The newsletter will keep participants 
up-to-date regarding the progress of the study and also touch upon health topics that are related 
to the study such as prevention of cardiovascular disease and cancer or current research 
findings. 
At the end of pill-taking, for the following two years, an annual questionnaire will be mailed to 
participants. The form will be similar to the conventional follow-up questionnaires, collecting 
information about potential risk factors for cancer and cardiovascular disease, use of 
medications, and medical history. The additional data will be used to address the main research 
questions and to examine new hypotheses regarding health promotion and disease prevention. 
 
E. ENDPOINT ASCERTAINMENT AND VALIDATION 
The primary cancer endpoint of VITAL will be total cancer incidence (excluding non-melanoma 
skin cancer), with cancer mortality and the individual sites of colorectal, breast, and prostate 
cancer as secondary endpoints. For CVD, the primary endpoint will be a composite endpoint of 
MI, stroke, and CVD mortality. Secondary endpoints will include a second composite endpoint 
adding revascularization procedures of CABG and PCI, as well as the individual endpoints of 
MI, stroke, revascularization, and CVD mortality. Additional endpoints of interest include heart 
failure, colorectal polyps, and periodontal disease. 
Participants who self-report one of the above study endpoints will be asked to sign a medical 
release form authorizing VITAL staff to obtain hospital/physician records. The request will be 
accompanied by a cover letter expressing sympathy for the diagnosis and explaining HIPAA 
guidelines and the scientific importance of record validation. Non-responders will be sent two 
additional requests for the medical release form. After the release is obtained, a copy will be 
sent to the treating hospital/physician. If there is no response within 1 month, a second request 
will be mailed, followed by a phone call.  
Beginning in the fall 2016, to increase the completeness of outcome information regarding MI, 
stroke, revascularization procedures of CABG and PCI, and cancer, information will be 
requested from Medicare (the Centers for Medicare and Medicaid Services). To obtain the 
information, participants will be identified using date of birth and social security number (if 
previously provided by the participant). The information will include medical diagnoses from 
hospitalizations, emergency room visits, as well as the names of procedures funded by 
Medicare. The information does not include test result or actual medical records. To obtain this 

 
13
specific information, participants will be contacted directly in order to gain their written 
permission for the release of this information (see above.) 
After all records are obtained, an Endpoints Committee of physicians, blinded to the randomized 
treatment assignment, will review the file and, using a defined protocol, will confirm or 
disconfirm the case. A cancer diagnosis will be confirmed with histologic or cytologic evidence. 
In the absence of these diagnostic tests, strong clinical evidence accompanied by radiologic 
evidence or laboratory markers (e.g., PSA levels) will be used to confirm cancer occurrence. 
The histologic type, grade, and stage of cancer will be recorded.70 MI will be confirmed using 
Joint European Society of Cardiology/American College of Cardiology Foundation/ American 
Heart Association/World Heart Federation (ESC/ACCF/AHA/WHF) Task Force for the 
Redefinition of Myocardial Infarction criteria.71 Stroke will be confirmed and categorized 
according to Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria.72 Death due to a 
cardiovascular cause will be confirmed by convincing evidence of a CVD event from all 
available sources, including death certificates, hospital records, autopsy, and observer accounts 
(for deaths outside the hospital). 
For reported deaths, the validation process will be similar. A condolence letter will be mailed to 
the family, requesting permission to obtain medical records and a copy of the death certificate. If 
the family does not provide the death certificate, a copy will be requested from the state vital 
records bureau where the participant died. The Endpoints Committee will review all records 
relevant to the death and assign an ICD code. At the end of the trial, we will search the National 
Death Index Plus (NDI-Plus) data base for known deaths for cases where we could not obtain 
records. NDI-Plus provides an ICD-coded cause of death based on death certificate information. 
We will also use the NDI to search for participants who become lost to follow-up. 
F. DATA MANAGEMENT 
Because participants will be followed solely by mail, our computing system is a critical feature of 
effective follow-up. This system, which was developed and fine-tuned in our previous trials, will 
track each participant’s stage in the study and level of participation. It will automatically generate 
letters, questionnaires, and phone call reminders at the appropriate times. Names, addresses, 
telephone numbers, participation status, and processing information will be kept up to date, and 
data from questionnaires, letters, and phone calls will be entered into the study database. When 
talking to a participant, study personnel will need ready access to identifying information, 
participation level, and the content of previous study-related telephone calls. However, it is also 
critical that these data be available only to authorized staff members. Our existing computer and 
security systems, which balance these considerations, will be used in VITAL.  
Questionnaire data will be optically scanned into the computer. The relevant software—
TELEform and Alchemy (Cardiff Software)—has been successfully used for several years in the 
WHS and PHS. Out-of-range, internally inconsistent, and unclear data will be reviewed by a 
verifier who will edit misread variables. Forms that cannot be scanned, and name and address 
changes, will be entered using traditional double-entry procedures. All data will undergo 
additional within-form and across-time checks to verify accuracy. The database will be 
maintained on a UNIX server. All data files will be backed up nightly, ensuring at least two 
current copies at all times. Each month, a set of data files will be taken off-site for long-term 
storage. 

 
14
Beginning in 2014, some study data will be collected via electronic forms sent to study 
participants via e-mail. The data will be collected and managed using REDCap electronic data 
capture tools. REDCap (Research Electronic Data Capture) is a free, secure, HIPAA-compliant 
web-based application hosted by Partners HealthCare Research Computing, Enterprise 
Research Infrastructure & Services (ERIS) group. Vanderbilt University with collaboration from a 
consortium of academic and non-profit institutional partners has developed this software toolset 
and workflow methodology for electronic collection and management of research study data. 
Our research team develops the data collection tools with planning assistance from Harvard 
Catalyst / The Harvard Clinical and Transitional Science Center EDC Support Staff. The 
development and testing process results in a well-planned data coillection strategy. REDCAp 
provides flexible features that can be used for a variety of research projects and provides an 
intuitive interface to enter data with real-time validation (automated data type and range 
checks). The system offers easy data manipulation with audit trails, reports for monitoring and 
querying participant records, and an automated export mechanism to common statistical 
packages (e.g., SAS). 
Participants’ names and contact information will be accessible only to staff members who need 
the information for their jobs. Endpoint and health-related questionnaire data will be stored in 
separate files from the processing data and will be accessible only to approved investigators 
and programmers. In these files, participants are identified only by study ID. 
VI. 
BIOSTATISTICAL ANALYSIS 
A. DATA ANALYSIS  
Analyses of treatment effects will be based on the intent-to-treat principle. The first analysis will 
compare baseline characteristics by randomized treatment assignment to ensure that balance 
was achieved by the randomization. Characteristics to be examined include known risk factors 
for cancer and CVD, including age; gender; race/ethnicity; BMI; smoking; alcohol use; physical 
activity; medical conditions such as hypertension, hyperlipidemia, diabetes, and family history of 
cancer and CVD; and baseline vitamin D and omega-3 fatty acid levels as assessed by dietary 
questionnaire in all participants and blood assays in a case-cohort subsample. The large 
sample size, as well as successful balance of known potential confounders, will provide 
assurance that unmeasured or unknown potential confounders will also be equally distributed 
across randomized treatment groups. 
In this 2x2 factorial design, the primary aim is to compare the main effects of intention-to-treat 
with vitamin D softgel capsules and with Omacor® fish oil on cancer and CVD. We will use the 
Cox proportional hazards model to allow for variable follow-up lengths73 and will estimate the 
hazard ratio for each intervention using indicators for treatment exposure, controlling for the 
second intervention, age, and gender. Because the cohort will consist of older individuals, 
competing risks due to deaths from other causes will be considered. The primary analysis will 
estimate the cause-specific hazard and the hazard ratio comparing intervention groups for each 
outcome of interest by censoring individuals with deaths due to competing causes. To estimate 
the cumulative incidence function, the subdistribution of each endpoint will be plotted over 
time.74,75 The alternative Fine and Gray approach76 models the effect of treatment on the 
subdistribution hazard or directly on the cumulative incidence function. While we will consider 
this and compare its fit to the model-free cumulative incidence curves,75 the proportional 
hazards approach will be our primary analysis. 

 
15
Similar analyses will be conducted for both cancer and CVD. For cancer, we will estimate the 
intervention effects on the primary endpoint of total cancer, as well as the secondary outcomes 
of breast, prostate, and colorectal cancer, and cancer mortality. For CVD we will estimate the 
effects on the primary composite endpoint of major CVD (MI, stroke, and cardiovascular 
mortality); on the secondary composite endpoint that additionally includes coronary 
revascularization; and on the individual components of MI, stroke, revascularization, and 
cardiovascular mortality. 
Beyond the primary analyses, we will conduct prespecified secondary analyses to examine 
effect modification by the other randomized intervention, by baseline risk factors, by compliance, 
and by time. We have a particular interest in exploring interactions between the interventions, as 
well as with the baseline plasma biomarkers of 25(OH)D for vitamin D and of EPA+DHA for fish 
oil. We hypothesize that the intervention effects may be larger among those with below-median 
baseline levels, and will examine treatment effects by quartiles of these biomarkers. The 
biomarker data will be analyzed as a case-cohort study using methods for proportional hazards 
regression.77 Because of the frequency matching by age and gender, appropriate stratum-
specific weighting of the observations will be employed.78  We also have a prior interest in the 
effects of the vitamin D intervention within groups defined by race/ethnicity and skin 
pigmentation, and by BMI. In addition, we will evaluate effect modification by age and gender, 
as well as by sunlight exposure, calcium and phosphorus intakes from the FFQ (as these 
nutrients affect vitamin D bioavailability79), and baseline risk factors for cancer and CVD. The 
latter interaction effects will be interpreted cautiously, as hypothesis generating. We will also 
examine as a prespecified secondary analysis the association among compliers, in which 
participants’ follow-up time will be censored when they are no longer compliant with taking at 
least two-thirds of their assigned active agents or placebos. Finally, we will examine whether 
treatment effects vary over time and duration of treatment by examining survival plots and 
interactions with time.  We will additionally conduct analyses for the primary endpoints of cancer 
and cardiovascular disease excluding events occurring in Year 1, as well as in Years 1 and 2, of 
follow-up. There may be a latent effect on both outcomes, depending on the mechanisms by 
which these agents act. 
We will also compare the incidence of potential side effects in the active v. placebo groups for 
each agent, including the incidence of kidney stones with vitamin D assignment (cf. WHI 
calcium-vitamin D findings44) and the incidence of GI symptoms, skin abnormalities, and 
bleeding with fish oil assignment (cf. JELIS findings41). 

 
16
B. POWER CALCULATIONS  
To calculate power, the following assumptions were made: (1) a 2x2 factorial trial in 12,500 men 
aged >50 and 12,500 women aged >55; (2) independent and equal allocation of participants to 
each treatment; (3) an age distribution based on that observed at baseline in our past trials for 
men aged ≥50 and women aged ≥55; (4) age-specific event rates based on the observed rates 
in the first 5 years of the PHS II for men and the WHS for women; (5) trial follow-up of 5 years, 
with little loss to follow-up as achieved in our past trials; and (6) compliance similar to that in 
published trials upon which our estimated rate ratio (RR) reductions are based. The cited 
reductions are thus the observed effects we would see in the trial. The corresponding ‘true’ RR 
is given assuming an average compliance of 80%. Power is given for a two-sided test using a 
logrank analysis80 with a significance level of 0.05. 
 
Cancer: Assuming that only one agent is effective, with 5 years of treatment and follow-up we 
will have 93% power to detect an observed RR of 0.85 for the primary endpoint of total cancer 
incidence (Table 1). For 
prostate cancer, we will 
have 93% power to 
detect a 25% risk 
reduction. For breast 
cancer, we will have 
86% power to detect a 
35% risk reduction. For 
colorectal cancer, we 
will have 77% power to 
detect a 35% risk 
reduction and 88% 
power to detect a 40% 
risk reduction. For 
cancer mortality, we will 
have 86% power to detect a 30% risk reduction. If both agents are effective in preventing cancer 
but act independently, power would be reduced slightly due to a somewhat smaller overall 
number of events. If the agents interact, however, power will be affected to the extent of the 
interaction. Should the agents act synergistically, power would increase, as illustrated in Table 2 
for the outcome of total cancer.  
For example, if the effect of each agent alone is a reduction of 10%, but in combination the 
effect is stronger, with an additional 10% decrease, the RR comparing the combined group to 
the all placebo group would be 0.73, versus 0.81 with additive effects (on the multiplicative 
scale). Power for the main effect of each agent would then increase to 88% (or higher with 
greater synergy). The 
trial would have power to 
detect moderate-to-large 
interactions, as shown in 
Table 2. Should the 
agents interact in a 
subadditive fashion, 
power would be reduced 

 
17
to the extent of the subadditivity. Power to explore effect modification by baseline blood levels of 
25(OH)D and EPA+DHA among those above and below the median level in the cohort will be 
lower because we anticipate that about 60% of participants will provide blood specimens.  
Power has been shown for a given observed reduction in risk, assuming compliance similar to 
that seen in current randomized trials such as the WHS and PHS. For total cancer, our power 
will be excellent with a 5-year follow-up even if compliance is lower. 
CVD: As shown in Table 3, 
assuming that only one 
agent is effective, with 5 
years of treatment and 
follow-up we will have 95% 
power to detect an observed 
RR of 0.80 for the primary 
composite endpoint of MI, 
stroke, and cardiovascular 
mortality. For the secondary 
composite endpoint of MI, 
stroke, cardiovascular 
mortality, and coronary 
revascularization, we will 
have over 99% power to 
detect a 20% reduction and 
93% power to detect a 15% 
reduction. With a RR of 0.70 
we will have 89% power for 
cardiovascular mortality, 
and we will have over 80% power to detect an RR of 0.75 for MI and stroke. If both agents are 
effective in preventing CVD but act independently, power would be reduced only slightly due to 
a smaller overall number of events. If the agents act synergistically, power would increase, as 
described for cancer. If the agents interact in subadditive fashion, power would be reduced to 
the extent of the subadditivity.  
VII. RISKS AND DISCOMFORTS 
 
The VITAL trial will test a dose of 2000 IU (50 µg) per day of vitamin D3 (cholecalciferol) softgel 
capsules and 840 mg/d of Omacor® EPA+DHA in VITAL. These doses provide the best 
balance of efficacy and safety, based on careful review of the literature and extensive 
consultation with numerous nutritional experts. We seek to obtain an adequate difference in 
vitamin D and EPA+DHA levels between the treatment and placebo groups to detect health 
benefits, and there appear to be few safety issues associated with these doses. For vitamin D, 
the selected dose is below the tolerable upper intake level (UL) for vitamin D3 (100 µg, or 4000 
IU) set by the Food and Nutrition Board of the Institute of Medicine (IOM). Additionally, because 
we will exclude from the trial persons who report supplemental vitamin D intakes >800 IU/d, the 
selected dose ensures that no participants will be taking a supplemental vitamin D dose above 
2800 IU, which is well below the 4000 IU tolerable upper intake level specified by the Institute of 
Medicine.2 To further ensure participants’ safety, we will exclude from the trial persons with a 
history of hypercalcemia, renal failure, cirrhosis, or sarcoidosis or other granulomatous disease, 
and will obtain calcium and PTH levels in a random subsample of participants. The dose of 

 
18
Omacor® fish oil used in this trial (840 mg/day) is consistent with guidelines set by health 
authorities who recommend up to 1 gram/day for cardio-protection. For fish oil, there are no 
specific exclusions necessary except for those currently taking the supplement and unwilling to 
forego use during the trial.   
Potential side effects of the study agents will be assessed on each follow-up questionnaire. For 
fish oil, these side effects include gastrointestinal (GI) symptoms (burping, stomach upset or 
pain, nausea, constipation, diarrhea), bleeding (any GI bleed, GI bleeding requiring transfusion, 
hematuria, easy bruising, epistaxis), skin rash, colds or upper respiratory tract infection, flu-like 
symptoms, and bad taste in mouth. A recent study reported that men with high blood levels of 
omega-3 fatty acids, which are found in fish and fish oil supplements, were more likely to 
develop prostate cancer than men with low blood levels of omega-3 fatty acids.81 However, 
because this was an observational study and not a randomized trial, the findings could be 
spurious, resulting from confounding bias. The VITAL trial will be collecting information on new 
diagnoses of prostate cancer, assessed on each follow-up questionnaire by self-report and 
confirmed on medical record review. For vitamin D, side effects include GI symptoms; and 
physician diagnosis of hypercalcemia or kidney stones, both of which are not expected to occur 
at the dose used in VITAL.2  
There is also the possibility of social/psychological risk that could result from inadvertent 
disclosure of confidential information from the questionnaires or blood tests. However, we have 
many safeguards in place to avoid this possibility, and we have never had an inadvertent breach 
of confidentiality in any of our trials.  
With regard to the risks of the study agents, information on potential side effects discussed 
above will be elicited on each follow-up questionnaire for monitoring by the Data and Safety 
Monitoring Board.  We will compare the incidence of potential side effects in the active v. 
placebo groups for each agent, including the incidence of kidney stones with vitamin D 
assignment and the incidence of GI symptoms, skin abnormalities, and bleeding with fish oil 
assignment. 
The potential risk of disclosure of confidential information is guarded against by maintaining 
completed questionnaires and blood test results in locked offices accessible by authorized 
personnel only. In questionnaire data files, participants are identified only by study ID. Subject 
identifiers are stored in separate computer files with password protection, and results will be 
presented only in the aggregate. In addition, employees involved with the proposed study will be 
asked to sign a form agreeing not to disclose any information to which they might have access, 
regardless of their personal perception about its confidentiality. Each employee of the study who 
has access to study data or has contact with subjects participates in an institutional (Brigham 
and Women’s Hospital) human subjects educational program, which consists of reviewing 
regulatory and informational documents pertaining to human subjects research; passing a test 
on ethical principles and regulations governing human subjects research; and signing a 
statement of commitment to the protection of human subjects.  Finally, all such employees are 
required to participate in HIPAA training 
 
VIII. POTENTIAL BENEFITS 
 
For the majority of participants, there will be few direct benefits from participating in this primary 
prevention study other than the awareness of being involved in a large endeavor to answer 
relevant and timely questions regarding the possible benefit of vitamin D and fish oil. During the 

 
19
trial itself, we will receive inquiries from the participants regarding both specific and general 
health concerns. We will respond to each of those questions, primarily directing participants to 
published sources or recommending that they see their local health provider who is familiar with 
their medical history.  
The purported health benefits of vitamin D and marine omega-3 fatty acids are receiving 
increasing attention in the medical literature and the popular press. Sales of vitamin D 
supplements and omega-3 fatty acid supplements at U.S. stores have increased substantially in 
recent years. However, definitive data on health benefits and risks of these agents are lacking. 
Findings from this large clinical trial will clarify the role of vitamin D and marine omega-3 fatty 
acid supplements in the primary prevention of cancer and CVD in men and women and will help 
guide individual decisions, clinical recommendations, and public health guidelines. 
 
IX. MONITORING AND QUALITY ASSURANCE 
 
A. SAFETY MONITORING 
 
An independent Data and Safety Monitoring Board (DSMB) will be assembled, consisting of 
experts in clinical trials, epidemiology, biostatistics, relevant clinical areas of cancer and CVD, 
and NIH representatives. The Physicians’ Health Study, the Women’s Health Study, and the 
Women’s Antioxidant and Folic Acid Cardiovascular Study have been monitored by the same 
DSMB since their inception. As two of these trials have ended and the third is in its last years, 
we have asked our DSMB members if they would be willing to also monitor VITAL. Four have 
accepted the invitation, and three new members have been added. The full 7-member DSMB 
has been approved by the NCI. 
The DSMB will annually examine the progress of the trial and the unblinded data on study 
endpoints and possible adverse effects to recommend continuation, alteration of study design, 
or early termination, as appropriate. Interim trial results will be assessed with the Haybittle-Peto 
rule,82,83 adjusting for multiple looks. In this method, interim results are compared to a z-score of 
3 standard deviations (p=0.0027) throughout the trial. The final results may then be interpreted 
as having close to nominal significance levels. This rule appropriately requires very strong 
evidence for early stopping, is more conservative than the Pocock84 and O’Brien-Fleming85 rules 
and the alpha-spending function,86 and can be conducted at convenient times without inducing 
statistical complexity.87 
While these rules are intended for the primary endpoints, the goal of VITAL is to assess the 
overall balance of benefits and risks of the two agents in the primary prevention of cancer and 
CVD. Thus, consideration will also be given to the secondary endpoints that are needed in the 
interpretation of overall results. In addition, the monitoring rules will serve solely as guidelines in 
decisions regarding continuation or stopping of treatment arms. All decisions must be made 
after examining the totality of evidence, including other trial data, on these agents. 
 
B. MONITORING OF SOURCE DATA AND DRUG DISPENSEMENT 
 
Redundancies will be built into the data processing systems to insure accurate recording of data 
and proper follow-up. All research forms will be scanned in and the data read by a character-
recognition software program (Teleform). Out-of-range, internally inconsistent, and unclear data 
will be reviewed by a verifier who will edit misread variables. Forms that cannot be scanned, 
and name and address changes, will be entered using traditional double-entry procedures. All 

 
20
data will undergo additional within-form and across-time checks to verify accuracy. Following 
data entry, all questionnaire responses that require additional follow-up for missing data, 
participant comments on the form or for endpoint validation will be manually reviewed to insure 
correct processing and accurate follow-up. Address changes received from the post office or 
from participants will be manually keyed by data entry personnel and the resultant files 
compared and verified. 
 
Great care will be taken to insure that participants receive their randomly assigned drugs. Upon 
receipt of drug shipments from the manufacturer, each of the four drug groups will be stored in 
segregated areas. A random sample of calendar packs will be pulled from each drug group and 
blindly tested to insure that the packaged contents match the study label. At the time of 
packaging for shipment to individual participants, listings will be divided according to drug group 
assignment and only one group will be packaged at a time. The packaging area will be cleaned 
and cleared prior to packaging and shipment of the next drug group. If drug packages are 
returned by the postal service as undeliverable or declined by a participant, the contents of the 
package will be blindly tested as an additional level of quality control. 
 
X. REFERENCES 
 
1. 
Davis CD, Hartmuller V, Freedman DM, Hartge P, Picciano MF, Swanson CA, Milner JA. 
Vitamin D and cancer: current dilemmas and future needs. Nutr Rev 2007; 65:S71-4. 
2. 
Institute of Medicine. Dietary Reference Intakes for Calcium and Vitamin D. Washington 
DC: National Academies Press, 2011. 
3. 
Giovannucci E. Strengths and limitations of current epidemiologic studies: vitamin D as a 
modifier of colon and prostate cancer risk. Nutr Rev 2007; 65:S77-9. 
4. 
Looker AC, Dawson-Hughes B, Calvo MS, Gunter EW, Sahyoun NR. Serum 25-
hydroxyvitamin D status of adolescents and adults in two seasonal subpopulations from 
NHANES III. Bone 2002; 30:771-7. 
5. 
Holick MF. Vitamin D deficiency. N Engl J Med 2007; 357:266-81. 
6. 
Harris SS. Vitamin D and African Americans. J Nutr 2006; 136:1126-9. 
7. 
Moore CE, Murphy MM, Holick MF. Vitamin D intakes by children and adults in the 
United States differ among ethnic groups. J Nutr 2005; 135:2478-85. 
8. 
Martins D, Wolf M, Pan D, Zadshir A, Tareen N, Thadhani R, Felsenfeld A, Levine B, 
Mehrotra R, Norris K. Prevalence of cardiovascular risk factors and the serum levels of 
25-hydroxyvitamin D in the United States: data from the Third National Health and 
Nutrition Examination Survey. Arch Intern Med 2007; 167:1159-65. 
9. 
Harris SS, Dawson-Hughes B. Reduced sun exposure does not explain the inverse 
association of 25-hydroxyvitamin D with percent body fat in older adults. J Clin 
Endocrinol Metab 2007; 92:3155-7. 
10. 
Hedley AA, Ogden CL, Johnson CL, Carroll MD, Curtin LR, Flegal KM. Prevalence of 
overweight and obesity among US children, adolescents, and adults, 1999-2002. JAMA 
2004; 291:2847-50. 
11. 
Ogden CL, Carroll MD, Curtin LR, McDowell MA, Tabak CJ, Flegal KM. Prevalence of 
overweight and obesity in the United States, 1999-2004. JAMA 2006; 295:1549-55. 
12. 
Kris-Etherton PM, Harris WS, Appel LJ. Fish consumption, fish oil, omega-3 fatty acids, 
and cardiovascular disease. Circulation 2002; 106:2747-57. 
13. 
NIH Office of Dietary Supplements, National Heart Lung and Blood Institute. Working 
Group Report on Future Clinical Research Directions on Omega-3 Fatty Acids and 
Cardiovascular Disease. June 2, 2004. Bethesda MD. Available at: 

 
21
http://www.nhlbi.nih.gov/meetings/workshops/omega-3/omega-3-rpt.htm Accessed on 
March 28, 2008. 
14. 
Stipp D. Fish-oil doses can be hard to swallow. Wall Street Journal: January 8, 2008; 
Page D1-D2. 
15. 
Gosnell M. Top 100 science stories of 2007: #8: Can vitamin D save your life? Discover: 
December 12, 2007. Available at http://discovermagazine.com/2008/jan/year-in-science-
2007/can-vitamin-d-save-your-life Accessed March 25, 2008. 
16. 
Guthrie C. The 10 biggest medical breakthroughs. #10: Benefits of vitamin D. Time: 
December 24, 2007. Available at: 
http://www.time.com/time/specials/2007/top10/article/0,30583,1686204_1686252_16903
93,00.html  Accessed March 25, 2008. 
17. 
Harvard Health Letter. The top 10 health stories of 2006. Harv Health Lett 2006; 32:1-3. 
18. 
Frisinger C. New year, new you: 8 simple rules for a healthier 2008. #1. Get more 
vitamin D. Fort Worth Star-Telegram (Texas): January 1, 2008; Page E1. 
19. 
Brody JE. An oldie vies for nutrient of the decade. New York Times: February 19, 2008; 
Page F7. 
20. 
Stampfer MJ. Vitamin D in the spotlight. Newsweek: December 11, 2006. Available at: 
http://www.newsweek.com/id/43998 Accessed March 25, 2008. 
21. 
Peng T. Don't forget your vitamins. Newsweek: March 24, 2008; Page 78. 
22. 
Rosenfeld I. We may need more vitamin D. Parade Magazine: January 29, 2006; Page 
15. 
23. 
Rhodes P. Vitamin D essentials: what you need to know about this important nutrient 
and where best to obtain it. Cooking Light 2008; 22:44-50. 
24. 
Foreman J. Is there any way to tell whether you are getting enough vitamin D? Boston 
Globe: February 4, 2008; Page C2.  
25. 
Harvard Health Letter. A more D-manding diet. Some experts say we should have a lot 
more vitamin D in our diets because it's protective against several diseases. Harv Health 
Lett 2007; 32:3.  
26. 
Simopoulos AP, Robinson J. The Omega Diet: The Lifesaving Nutritional Program 
Based on the Diet of the Island of Crete. New York: HarperCollins, 1999. 
27. 
Sears B. The Omega Rx Zone: The Miracle of the New High-Dose Fish Oil. New York: 
HarperCollins, 2004. 
28. 
Kaufman M. Fish oil may aid against manic depression. Washington Post, 1999:Z07. 
29. 
Foreman J. Fatty acid imbalance hurts our health. Boston Globe: February 10, 2004. 
30. 
Bakalar N. Fish oil linked to lower Alzheimer's risk. New York Times: November 14, 
2006. 
31. 
Munoz SS. Shopping around: DHA-enhanced foods. Wall Street Journal: March 27, 
2008; Page D2. 
32. 
Canadian Cancer Society. Canadian Cancer Society Announces Vitamin D 
Recommendation. Available at: 
http://www.cancer.ca/ccs/internet/mediareleaselist/0,,3172_1613121606_1997621989_l
angId-en.html. Accessed May 6, 2008. 
33. 
Vieth R, Bischoff-Ferrari H, Boucher BJ, Dawson-Hughes B, Garland CF, Heaney RP, 
Holick MF, Hollis BW, Lamberg-Allardt C, McGrath JJ, Norman AW, Scragg R, Whiting 
SJ, Willett WC, Zittermann A. The urgent need to recommend an intake of vitamin D that 
is effective. Am J Clin Nutr 2007; 85:649-50. 
34. 
Davis CD, Dwyer JT. The "sunshine vitamin": benefits beyond bone? J Natl Cancer Inst 
2007; 99:1563-5. 
35. 
Bardia A, Tleyjeh IM, Cerhan JR, Sood AK, Limburg PJ, Erwin PJ, Montori VM. Efficacy 
of antioxidant supplementation in reducing primary cancer incidence and mortality: 
systematic review and meta-analysis. Mayo Clin Proc 2008; 83:23-34. 

 
22
36. 
Mosca L, Banka CL, Benjamin EJ, Berra K, Bushnell C, Dolor RJ, Ganiats TG, Gomes 
AS, Gornik HL, Gracia C, Gulati M, Haan CK, Judelson DR, Keenan N, Kelepouris E, 
Michos ED, Newby LK, Oparil S, Ouyang P, Oz MC, Petitti D, Pinn VW, Redberg RF, 
Scott R, Sherif K, Smith SC, Jr., Sopko G, Steinhorn RH, Stone NJ, Taubert KA, Todd 
BA, Urbina E, Wenger NK. Evidence-based guidelines for cardiovascular disease 
prevention in women: 2007 update. Circulation 2007; 115:1481-501. 
37. 
Kris-Etherton PM, Lichtenstein AH, Howard BV, Steinberg D, Witztum JL. Antioxidant 
vitamin supplements and cardiovascular disease. Circulation 2004; 110:637-41. 
38. 
Lee IM, Cook NR, Gaziano JM, Gordon D, Ridker PM, Manson JE, Hennekens CH, 
Buring JE. Vitamin E in the primary prevention of cardiovascular disease and cancer: the 
Women's Health Study: a randomized controlled trial. JAMA 2005; 294:56-65. 
39. 
Miller ER, 3rd, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E. Meta-
analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann 
Intern Med 2005; 142:37-46. 
40. 
Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C. Mortality in randomized 
trials of antioxidant supplements for primary and secondary prevention: systematic 
review and meta-analysis. JAMA 2007; 297:842-57. 
41. 
Yokoyama M, Origasa H, Matsuzaki M, Matsuzawa Y, Saito Y, Ishikawa Y, Oikawa S, 
Sasaki J, Hishida H, Itakura H, Kita T, Kitabatake A, Nakaya N, Sakata T, Shimada K, 
Shirato K. Effects of eicosapentaenoic acid on major coronary events in 
hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint 
analysis. Lancet 2007; 369:1090-8. 
42. 
Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico. Dietary 
supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial 
infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della 
Sopravvivenza nell'Infarto miocardico. Lancet 1999; 354:447-55. 
43. 
Lappe JM, Travers-Gustafson D, Davies KM, Recker RR, Heaney RP. Vitamin D and 
calcium supplementation reduces cancer risk: results of a randomized trial. Am J Clin 
Nutr 2007; 85:1586-91. 
44. 
Wactawski-Wende J, Kotchen JM, Anderson GL, Assaf AR, Brunner RL, O'Sullivan MJ, 
Margolis KL, Ockene JK, Phillips L, Pottern L, Prentice RL, Robbins J, Rohan TE, Sarto 
GE, Sharma S, Stefanick ML, Van Horn L, Wallace RB, Whitlock E, Bassford T, 
Beresford SA, Black HR, Bonds DE, Brzyski RG, Caan B, Chlebowski RT, Cochrane B, 
Garland C, Gass M, Hays J, Heiss G, Hendrix SL, Howard BV, Hsia J, Hubbell FA, 
Jackson RD, Johnson KC, Judd H, Kooperberg CL, Kuller LH, LaCroix AZ, Lane DS, 
Langer RD, Lasser NL, Lewis CE, Limacher MC, Manson JE. Calcium plus vitamin D 
supplementation and the risk of colorectal cancer. N Engl J Med 2006; 354:684-96. 
45. 
Hathcock JN, Shao A, Vieth R, Heaney R. Risk assessment for vitamin D. Am J Clin 
Nutr 2007; 85:6-18. 
46. 
Lang JM, Buring JE, Rosner B, Cook N, Hennekens CH. Estimating the effect of the run-
in on the power of the Physicians' Health Study. Stat Med 1991; 10:1585-93. 
47. 
Talwar SA, Aloia JF, Pollack S, Yeh JK. Dose response to vitamin D supplementation 
among postmenopausal African American women. Am J Clin Nutr 2007; 86:1657-62. 
48. 
Rimm EB, Giovannucci EL, Stampfer MJ, Colditz GA, Litin LB, Willett WC. 
Reproducibility and validity of an expanded self-administered semiquantitative food 
frequency questionnaire among male health professionals. Am J Epidemiol 1992; 
135:1114-26. 
49. 
Feskanich D, Rimm EB, Giovannucci EL, Colditz GA, Stampfer MJ, Litin LB, Willett WC. 
Reproducibility and validity of food intake measurements from a semiquantitative food 
frequency questionnaire. J Am Diet Assoc 1993; 93:790-6. 

 
23
50. 
Salvini S, Hunter DJ, Sampson L, Stampfer MJ, Colditz GA, Rosner B, Willett WC. Food-
based validation of a dietary questionnaire: the effects of week-to-week variation in food 
consumption. Int J Epidemiol 1989; 18:858-67. 
51. 
Jacques PF, Sulsky SI, Sadowski JA, Phillips JC, Rush D, Willett WC. Comparison of 
micronutrient intake measured by a dietary questionnaire and biochemical indicators of 
micronutrient status. Am J Clin Nutr 1993; 57:182-9. 
52. 
Feskanich D, Willett WC, Colditz GA. Calcium, vitamin D, milk consumption, and hip 
fractures: a prospective study among postmenopausal women. Am J Clin Nutr 2003; 
77:504-11. 
53. 
Hunter DJ, Rimm EB, Sacks FM, Stampfer MJ, Colditz GA, Litin LB, Willett WC. 
Comparison of measures of fatty acid intake by subcutaneous fat aspirate, food 
frequency questionnaire, and diet records in a free-living population of US men. Am J 
Epidemiol 1992; 135:418-27. 
54. 
Bischoff-Ferrari HA, Giovannucci E, Willett WC, Dietrich T, Dawson-Hughes B. 
Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health 
outcomes. Am J Clin Nutr 2006; 84:18-28. 
55. 
Dawson-Hughes B, Heaney RP, Holick MF, Lips P, Meunier PJ, Vieth R. Estimates of 
optimal vitamin D status. Osteoporos Int 2005; 16:713-6. 
56. 
Giovannucci E, Liu Y, Rimm EB, Hollis BW, Fuchs CS, Stampfer MJ, Willett WC. 
Prospective study of predictors of vitamin D status and cancer incidence and mortality in 
men. J Natl Cancer Inst 2006; 98:451-9. 
57. 
Aloia JF, Patel M, DiMaano R, Li-Ng M, Talwar SA, Mikhail M, Pollack S, Yeh  
            JK. Vitamin D intake to attain a desired serum 25-hydroxyvitamin D concentration. Am J 
Clin Nutr 2008;87:1952-1958 
58. 
Harris WS. International recommendations for consumption of long-chain omega-3 fatty 
acids. J Cardiovasc Med (Hagerstown) 2007; 8 Suppl 1:S50-2. 
59. 
Lee JH, O'Keefe JH, Lavie CJ, Marchioli R, Harris WS. Omega-3 fatty acids for 
cardioprotection. Mayo Clin Proc 2008; 83:324-32. [Docket No. 86G-0289], 1997. 
60. 
Agency for Healthcare Research and Quality. Effects of Omega-3 Fatty Acids on 
Cardiovascular Disease. Evidence Report/Technology Assessment Number 94. AHRQ 
Pub. No. 04-E009-1. 2004. Available at: 
http://www.ahrq.gov/clinic/epcsums/o3cardsum.htm Accessed March 28, 2008. 
61. 
Simopoulos AP. Evolutionary aspects of diet, the omega-6/omega-3 ratio and genetic 
variation: nutritional implications for chronic diseases. Biomed Pharmacother 2006; 
60:502-7. 
62. 
Kris-Etherton PM, Taylor DS, Yu-Poth S, Huth P, Moriarty K, Fishell V, Hargrove RL, 
Zhao G, Etherton TD. Polyunsaturated fatty acids in the food chain in the United States. 
Am J Clin Nutr 2000; 71:179S-88S. 
63. 
Harris WS. The omega-6/omega-3 ratio and cardiovascular disease risk: uses and 
abuses. Curr Atheroscler Rep 2006; 8:453-9. 
64. 
Stanley JC, Elsom RL, Calder PC, Griffin BA, Harris WS, Jebb SA, Lovegrove JA, Moore 
CS, Riemersma RA, Sanders TA. UK Food Standards Agency Workshop Report: the 
effects of the dietary n-6:n-3 fatty acid ratio on cardiovascular health. Br J Nutr 2007; 
98:1305-10. 
65. 
Harris WS, Poston WC, Haddock CK. Tissue n-3 and n-6 fatty acids and risk for 
coronary heart disease events. Atherosclerosis 2007; 193:1-10. 
66. 
Mozaffarian D, Ascherio A, Hu FB, Stampfer MJ, Willett WC, Siscovick DS, Rimm EB. 
Interplay between different polyunsaturated fatty acids and risk of coronary heart 
disease in men. Circulation 2005; 111:157-64. 
67. 
Department of Health and Human Services, U.S. Food and Drug Administration. 
Substances affirmed as generally recognized as safe: menhaden oil. Federal Register. 

 
24
June 5, 1997. Vol 62, No 108: pp 30751-30757. 21 CFR Part 184 [Docket No. 86G-
0289], 1997. 
68. 
Bays HE. Safety considerations with omega-3 fatty acid therapy. Am J Cardiol 2007; 
99:35C-43C. 
69. 
Satterfield S, Greco PJ, Goldhaber SZ, Stampfer MJ, Swartz SL, Stein EA, Kaplan L, 
Hennekens CH. Biochemical markers of compliance in the Physicians' Health Study. Am 
J Prev Med 1990; 6:290-4. 
70. 
Fritz AG, Percy C, Jack A, Shanmugaratham K, Sobin LH, Parkin MD, Whelan S. 
International Classification of Diseases for Oncology (ICD-O), Third Edition. Geneva: 
World Health Organization, 2000. 
71. 
Thygesen K, Alpert JS, White HD, Joint ESC/ACCF/AHA/WHF Task Force for the 
Redefinition of Myocardial Infarction. Universal definition of myocardial infarction. 
Circulation 2007; 116:2634-53. 
72. 
Adams HP, Jr., Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, Marsh EE, 3rd. 
Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter 
clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke 1993; 24:35-
41. 
73. 
Cox DR. Regression models and life-tables. J Royal Statist Soc B 1972; 34:187-220. 
74. 
Gooley TA, Leisenring W, Crowley J, Storer BE. Estimation of failure probabilities in the 
presence of competing risks: new representations of old estimators. Stat Med 1999; 
18:695-706. 
75. 
Putter H, Fiocco M, Geskus RB. Tutorial in biostatistics: competing risks and multi-state 
models. Stat Med 2007; 26:2389-430. 
76. 
Fine JP, Gray RJ. A proportional hazards model for the subdistribution of a competing 
risk. J Am Stat Assoc 1999; 94:496-509. 
77. 
Barlow WE. Robust variance estimation for the case-cohort design. Biometrics 1994; 
50:1064-72. 
78. 
Borgan O, Langholz B, Samuelsen SO, Goldstein L, Pogoda J. Exposure stratified case-
cohort designs. Lifetime Data Anal 2000; 6:39-58. 
79. 
Holick MF. Vitamin D for health and in chronic kidney disease. Semin Dial 2005; 18:266-
75 
80. 
Freedman LS. Tables of the number of patients required in clinical trials using the 
logrank test. Stat Med 1982; 1:121-9. 
81. 
Brasky TM, Darke AK, Song X, et al. Plasma phospholipid fatty acids and prostate 
cancer risk in the SELECT trial. J Natl Cancer Inst 2013; 105:1132-41. 
82. 
Haybittle JL. Repeated assessment of results in clinical trials of cancer treatment. Br J 
Radiol 1971; 44:793-7. 
83. 
Peto R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard SV, Mantel N, McPherson 
K, Peto J, Smith PG. Design and analysis of randomized clinical trials requiring 
prolonged observation of each patient. I. Introduction and design. Br J Cancer 1976; 
34:585-612. 
84. 
Geller NL, Pocock SJ. Interim analyses in randomized clinical trials: ramifications and 
guidelines for practitioners. Biometrics 1987; 43:213-23. 
85. 
O'Brien PC, Fleming TR. A multiple testing procedure for clinical trials. Biometrics 1979; 
35:549-56. 
86. 
DeMets DL, Lan KK. Interim analysis: the alpha spending function approach. Stat Med 
1994; 13:1341-52. 
87. 
Pocock SJ. Current controversies in data monitoring for clinical trials. Clin Trials 2006; 
3:513-21. 
 

August 21, 2018 
 
The VITamin D and OmegA-3 TriaL (VITAL): Summary of Changes to the Protocol:   
 
Our initial IRB application, submitted in June 2009, contained a draft protocol. 
 
January 28, 2010 (v. 2) was the date that the first full protocol was approved by the IRB. A copy of this 
protocol is provided. 
 
January 24, 2011 (v. 3) 
• 
Removed history of kidney stones as an exclusion criterion 
• 
Updated information on the RDA for vitamin D and calcium based on the Nov 2010 IOM report 
• 
For blood draw, replaced wording of “1 tube sodium citrate” with “1 serum tube” 
• 
Specified that we expected to mail recruitment materials to 3.0 million people 
• 
Specified that we would use an abbreviated version of the Willett Diet Assessment 
Questionnaire 
• 
Specified that the vitamin D agents will be in the form of a soft-gel capsule 
• 
Specified the omega-3 fatty acid agent as Omacor 
 
March 31, 2011 (v. 4) 
• 
Removed the education restriction that participants must have at least a high school education 
 
June 8, 2011 (v.5) 
• 
Eligible age range was lowered to 50 years or older for males and 55 or older for females, to 
reflect the younger age of development of heart disease and cancer among underrepresented 
minorities, and thus facilitate minority recruitment. 
 
December 1, 2011 (v. 6) 
• 
Volume of recruitment mailings was adjusted to 6 million people 
• 
Angina was deleted as an exclusion 
• 
Use of vitamin D analogs was added as an exclusion 
• 
Recruitment via local health fairs was added as a recruitment strategy 
• 
Added kidney failure or dialysis, parathyroid disease, cirrhosis or severe liver disease, use of 
anticoagulants as additional safety exclusions 
• 
Added that staff will contact willing participants to schedule a home visit or clinic visit for 
collection of blood sample 
• 
Specified that blood assays would be performed at Quest Diagnostics (other labs were deleted) 
• 
Revised power calculations based on lowering the eligibility age range on 6/8/11.   
 
January 6, 2012 (v. 7) 
• 
Add “burping” as a side effect of fish oil 
• 
Removed atrial fibrillation as a side effect of fish oil due to inconsistent evidence. 
 
December 3, 2012 (v. 8) 
• 
Specified that study will be conducted among 25,000 men and women 
• 
Specified that the mailing company will conduct recruitment mailings in volume of 0.5-1 million 
per drop 

• 
The number of willing and eligible persons to receive screening questionnaire (V-2) was 
projected to be 160,000 
• 
Specified that non-responders to run-in follow-up questionnaire (V-3) will receive up to 3 
requests for data 
• 
Specified that toward the end of the enrollment phase, as a mechanism to enhance minority 
representation in the trial, we will impose a stricter compliance standard on white, non-Hispanic 
enrollees who live outside the 60-mile range for CTSC recruitment: not eligible if a blood sample 
is not returned and missed more than 5 days of run-in placebo pills in past month 
• 
Contacting the “not sure” and “no” respondents to question about providing a blood sample to 
inform them that we can arrange a home visit if they wanted 
• 
Study guidelines amended to encourage randomized participants to inform their physicians 
about their participation in the trial if they receive a post-randomization diagnosis of 
hypercalcemia, sarcoidosis, or other safety issues 
• 
Provided new power calculations based upon a study population of 25,000 
 
April 1, 2013 (v. 9) 
• 
Blood sample analyses now to include measures of biomarkers related to CVD (cholesterol 
lipoprotein sub-fractions) – to be conducted at Atherotech Diagnostic Labs 
• 
Specified that non-responders to annual follow-up questionnaires will receive up to 5 requests 
for information 
 
September 10, 2013 (v. 10) 
• 
Added information about the SELECT Study results (observational association between omega-3 
fatty acids and risk of prostate cancer) to the “Background and Significance” section of protocol 
 
September 17, 2013 (v. 11) 
• 
Specified that the projected randomized population would be a maximum of 26,500 
• 
Added biochemical assays of blood specimens of metabolomic/lipidomic biomarkers for 
baseline and follow-up samples 
 
December 5, 2013 (v. 12) 
• 
Specified that internet sources will be used to assist in the tracking (i.e., search for new address 
and phone numbers) of participants potentially lost to follow-up 
 
December 17, 2013 (v. 13) 
• 
Added measurement of plasma levels of PCSK9 and plasma-based genotypes for PCSK9 
 
March 19, 2014 (v. 14) 
• 
Specified an increase in the target for optional follow-up blood sample from 2,000 to 6,000. 
Deletion of “random” to describe selection of follow-up samples, as participants asked to 
provide the samples will be those scheduled for an ancillary blood draw at a home visit. 
 
May 21, 2014 (v. 15) 
• 
Specified that participants will be given the option of completing their annual questionnaire via 
the Internet. 
 
June 19, 2014 (v. 16) 

• 
Specified the planned collection of the follow-up Diet Assessment at 3 years instead of 2 years 
• 
Added as a tertiary aim whether vitamin D or omega-3 supplementation reduces the risks of 
heart failure (ancillary study) 
 
November 11, 2015 (v. 17) 
• 
Specified that the follow-up Diet Assessment would be conducted at 4.5 years instead of 3 years 
• 
Added to “Endpoint Ascertainment” section:  Additional endpoints of interest include heart 
failure, colorectal polyps, and periodontal disease. 
 
January 6, 2016 (v. 18) 
• 
Added biochemical assays: measures of metabolomics and lipidomic biomarkers to be done at 
LabCorp in addition to the metabolomics laboratory at UCSD 
 
April 29, 2016 (v. 19) 
• 
Deleted reference to an additional Diet Assessment at conclusion of the trial (to minimize 
participant burden). 
 
September 12, 2016 (v. 20) 
• 
To increase completeness of ascertainment of study outcome data (for MI, stroke, 
revascularization, and cancer), specified that Medicare/CMS linkage would be done. However, 
participants will be directly asked to provide authorization to obtain medical records to validate 
outcomes ascertained from Medicare/CMS. 
 
January 27, 2017 (v. 21) 
• 
Specified the addition of biochemical assays: albumin, creatinine, liver function, lipid tests, 
cholesterol efflux, epigenetic biomarkers. 
 
September 13, 2017 (v. 22) 
• 
Specified that, at the conclusion of the randomized trial, we will send participants two additional 
annual questionnaires to collect supplemental study data during the post-intervention phase. 
 
November 30, 2017 (v. 23) is the current protocol.  A copy is provided. 
• 
Clarified that we will additionally conduct analyses excluding events occurring in year 1 and 
years 1-2 of follow-up (our original protocol on 1/28/10 [v.2] specified: “Finally, we will examine 
whether treatment effects vary over time and duration of treatment by examining survival plots 
and interactions with time. There may be a latent effect on cancer incidence, depending on the 
stage of carcinogenesis during which these agents act.”  In the 11/30/17 protocol, we formalized 
this as analyses excluding events during year 1 and years 1-2 of follow-up.) 
• 
Data analysis section amplified:  specified sensitivity analyses by compliance, with participants’ 
follow-up time censored when they are no longer compliant with taking at least two-thirds of 
their assigned active agents or placebos (as in our previous trials). 
 
 
NOTE:  VITAL investigators were unblinded to randomization treatment codes on December 6, 2017 
prior to a teleconference with VITAL DSMB members.  All of the above protocol changes were made 
PRIOR to the investigator unblinding on December 6, 2017. The intervention phase of VITAL ended on 
December 31, 2017. 

 
1
 
 PROTOCOL FOR THE VITAL TRIAL  
January 28, 2010 
 
 
 
VI. 
BIOSTATISTICAL ANALYSIS 
A. DATA ANALYSIS  
Analyses of treatment effects will be based on the intent-to-treat principle. The first analysis will 
compare baseline characteristics by randomized treatment assignment to ensure that balance 
was achieved by the randomization. Characteristics to be examined include known risk factors 
for cancer and CVD, including age; gender; race/ethnicity; BMI; smoking; alcohol use; physical 
activity; medical conditions such as hypertension, hyperlipidemia, diabetes, and family history of 
cancer and CVD; and baseline vitamin D and omega-3 fatty acid levels as assessed by dietary 
questionnaire in all participants and blood assays in a case-cohort subsample. The large 
sample size, as well as successful balance of known potential confounders, will provide 
assurance that unmeasured or unknown potential confounders will also be equally distributed 
across randomized treatment groups. 
In this 2x2 factorial design, the primary aim is to compare the main effects of intention-to-treat 
with vitamin D and with fish oil on cancer and CVD. We will use the Cox proportional hazards 
model to allow for variable follow-up lengths76 and will estimate the hazard ratio for each 
intervention using indicators for treatment exposure, controlling for the second intervention, age, 
and gender. Because the cohort will consist of older individuals, competing risks due to deaths 
from other causes will be considered. The primary analysis will estimate the cause-specific 
hazard and the hazard ratio comparing intervention groups for each outcome of interest by 
censoring individuals with deaths due to competing causes. To estimate the cumulative 
incidence function, the subdistribution of each endpoint will be plotted over time.77, 78 The 
alternative Fine and Gray approach79 models the effect of treatment on the subdistribution 
hazard or directly on the cumulative incidence function. While we will consider this and compare 
its fit to the model-free cumulative incidence curves,78 the proportional hazards approach will be 
our primary analysis. 
Similar analyses will be conducted for both cancer and CVD. For cancer, we will estimate the 
intervention effects on the primary endpoint of total cancer, as well as the secondary outcomes 
of breast, prostate, and colorectal cancer, and cancer mortality. For CVD we will estimate the 
effects on the primary composite endpoint of major CVD (MI, stroke, and cardiovascular 
mortality); on the secondary composite endpoint that additionally includes coronary 
revascularization; and on the individual components of MI, stroke, revascularization, and 
cardiovascular mortality. 
Beyond the primary analyses, we will examine effect modification by the other randomized 
intervention, by baseline risk factors, and by time. We have a particular interest in exploring 
interactions between the interventions, as well as with the baseline plasma biomarkers of 
25(OH)D for vitamin D and of EPA+DHA for fish oil. We hypothesize that the intervention effects 
may be larger among those with below-median baseline levels, and will examine treatment 
effects by quartiles of these biomarkers. The biomarker data will be analyzed as a case-cohort 
study using methods for proportional hazards regression.80 Because of the frequency matching 
by age and gender, appropriate stratum-specific weighting of the observations will be 
employed.81 We also have a prior interest in the effects of the vitamin D intervention within 

 
2
groups defined by race/ethnicity and skin pigmentation, and by BMI. In addition, we will evaluate 
effect modification by age and gender, as well as by sunlight exposure, calcium and phosphorus 
intakes from the FFQ (as these nutrients affect vitamin D bioavailability82), and baseline risk 
factors for cancer and CVD. The latter interaction effects will be interpreted cautiously, as 
hypothesis generating. Finally, we will examine whether treatment effects vary over time and 
duration of treatment by examining survival plots and interactions with time. There may be a 
latent effect on cancer incidence, depending on the stage of carcinogenesis during which these 
agents act. 
We will also compare the incidence of potential side effects in the active v. placebo groups for 
each agent, including the incidence of kidney stones with vitamin D assignment (cf. WHI 
calcium-vitamin D findings44) and the incidence of GI symptoms, skin abnormalities, and 
bleeding with fish oil assignment (cf. JELIS findings41). 
B. POWER CALCULATIONS  
To calculate power, the following assumptions were made: (1) a 2x2 factorial trial in 10,000 men 
aged >60 and 10,000 women aged >65; (2) independent and equal allocation of participants to 
each treatment; (3) an age distribution based on that observed at baseline in our past trials for 
men aged ≥60 and women aged ≥65; (4) age-specific event rates based on the observed rates 
in the first 5 years of the PHS II for men and the WHS for women; (5) trial follow-up of 5 years, 
with little loss to follow-up as achieved in our past trials; and (6) compliance similar to that in 
published trials upon which our estimated rate ratio (RR) reductions are based. The cited 
reductions are thus the observed effects we would see in the trial. The corresponding ‘true’ RR 
is given assuming an average compliance of 80%. Power is given for a two-sided test using a 
logrank analysis83 with a significance level of 0.05. 
Cancer: Assuming that 
only one agent is 
effective, with 5 years of 
treatment and follow-up 
we will have 91% power 
to detect an observed 
RR of 0.85 for the 
primary endpoint of total 
cancer incidence (Table 
1). For prostate cancer, 
we will have 88% power 
to detect a 25% risk reduction. For breast cancer, we will have 81% power to detect a 35% risk 
reduction. For colorectal cancer, we will have 78% power to detect a 35% risk reduction and 
88% power to detect a 40% risk reduction. For cancer mortality, we will have 87% power to 
detect a 30% risk reduction. If both agents are effective in preventing cancer but act 
independently, power would be reduced slightly due to a somewhat smaller overall number of 
events. If the agents interact, however, power will be affected to the extent of the interaction. 
Should the agents act synergistically, power would increase, as illustrated in Table 2 for the 
outcome of total cancer.  
For example, if the effect of each agent alone is a reduction of 10%, but in combination the 
effect is stronger, with an additional 10% decrease, the RR comparing the combined group to 
the all placebo group would be 0.73, versus 0.81 with additive effects (on the multiplicative 
Table 1. Power for effects of a single agent on cancer in a factorial trial of 10,000 men 
aged≥60 and 10,000 women aged≥65 with 5 years of follow-up 
Observed 
RR† 
True 
RR 
Total   
Cancer 
Cancer 
Mortality 
Colorectal 
Cancer 
Breast 
Cancer 
(Women) 
Prostate 
Cancer 
(Men) 
0.90 
0.875 
58.7 
- 
- 
- 
- 
0.85 
0.812 
91.1 
- 
- 
- 
- 
0.80 
0.750 
99.4 
52.0 
31.8 
34.6 
70.1 
0.75 
0.687 
>99.9 
72.2 
47.0 
50.9 
88.3 
0.70 
0.625 
>99.9 
87.3 
63.1 
67.5 
96.9 
0.65 
0.560 
>99.9 
95.6 
77.6 
81.5 
99.5 
0.60 
0.500 
>99.9 
98.9 
88.4 
91.2 
>99.9 
†Observed RR=intent-to-treat RR, including noncompliant participants. Compliance assumed 
to be 80%. True RR=that with perfect compliance. 

 
3
scale). Power for the main effect of each agent would then increase to 86% (or higher with 
greater synergy). The trial would have power to detect moderate-to-large interactions, as shown 
in Table 2. Should the agents interact in a subadditive fashion, power would be reduced to the 
extent of the subadditivity. Power to explore effect modification by baseline blood levels of 
25(OH)D and EPA+DHA among those above and below the median level in the cohort will be 
lower because we anticipate that about 60% of participants will provide blood specimens. We 
will have about 90% power to detect an interaction of size 0.6. 
Power has been shown for a given observed reduction in risk, assuming compliance similar to 
that seen in current randomized trials such as the WHS and PHS. For total cancer, our power 
will be excellent with a 5-year follow-up even if compliance is lower. For example, if the true 
reduction in risk is 20%, the observed RR corresponding to compliance of 80% is 84%, and we 
have 94% power to detect this level of effect. If compliance is only 75%, the observed RR would 
be 0.85, for which we would have 91% power. Finally, if compliance is only 70%, the observed 
RR would be 0.86, and we would still have 87% power to detect an effect of this magnitude. 
 
 
 
 
 
 
 
 
 
CVD: As shown in Table 3, assuming that only one agent is effective, with 5 years of treatment 
and follow-up we will have 92% power to detect an observed RR of 0.80 for the primary 
composite endpoint of MI, stroke, and cardiovascular mortality. For the secondary composite 
endpoint of MI, stroke, cardiovascular mortality, and coronary revascularization, we will have 
99% power to detect a 20% reduction and 90% power to detect a 15% reduction. With a RR of 
0.70 we will have 87% power for cardiovascular mortality, 88% power for MI, and 90% power for 
stroke. If both agents are effective in preventing CVD but act independently, power would be 
reduced only slightly due to a smaller overall number of events. If the agents act synergistically, 
power would increase, as described for cancer. If the agents interact in subadditive fashion, 
power would be reduced to the extent of the subadditivity.  
Table 2. Power for interaction effects on total cancer in a factorial trial of 
10,000 men aged ≥60 and 10,000 women aged ≥65 with 5 years of follow-up 
RR 
Single agent† 
Interaction† 
RR  
Both agents 
Power 
Main Effect      Interaction 
0.90 
1.0 
0.81 
56.3 
 
 
0.9 
0.73 
86.3 
 
 
0.8 
0.65 
98.0 
57.9 
 
0.7 
0.57 
99.9 
92.1 
0.85 
1.0 
0.72 
88.7 
 
 
0.9 
0.65 
98.1 
 
 
0.8 
0.58 
99.8 
55.0 
 
0.7 
0.51 
>99.9 
90.2 
0.80 
1.0 
0.64 
98.8 
 
 
0.9 
0.58 
99.9 
 
 
0.8 
0.51 
>99.9 
52.0 
 
0.7 
0.45 
>99.9 
87.9 
†RR = intent-to-treat RR, including noncompliant participants (assuming 80% 
compliance). Represents the effect among those not assigned to the other 
intervention and assumes the same effect for both agents. The interaction is the 
RR for the combined group divided by the product of risks for the two separate 
groups—i.e., RRint=RRboth/(RR vit D alone* RR fish oil alone). An interaction=1 
implies additive effects (no interaction). 

 
4
 
 
 
 
 
 
 
For total CVD, our power would be somewhat lower—but still adequate—with more limited 
compliance. If the true effect is a 25% reduction, power would be 92% if compliance is 80% 
(observed RR=0.80), 89% if compliance is 75% (observed RR=0.813), and 84% if compliance is 
70% (observed RR=0.825). To achieve 80% power for CVD, we would need an observed RR of 
at least 0.83, which corresponds to a true reduction of 21% under 80% compliance and 24% 
under 70% compliance. 
 
IX. MONITORING AND QUALITY ASSURANCE 
 
A. SAFETY MONITORING 
 
An independent Data and Safety Monitoring Board (DSMB) will be assembled, consisting of 
experts in clinical trials, epidemiology, biostatistics, relevant clinical areas of cancer and CVD, 
and NIH representatives. The Physicians’ Health Study, the Women’s Health Study, and the 
Women’s Antioxidant and Folic Acid Cardiovascular Study have been monitored by the same 
DSMB since their inception. As two of these trials have ended and the third is in its last years, 
we will ask our DSMB members if they would be willing to also monitor VITAL. If they are not 
able to do so, we will consult with them in assembling a new DSMB.  
The DSMB will annually examine the progress of the trial and the unblinded data on study 
endpoints and possible adverse effects to recommend continuation, alteration of study design, 
or early termination, as appropriate. Interim trial results will be assessed with the Haybittle-Peto 
rule,84, 85 adjusting for multiple looks. In this method, interim results are compared to a z-score of 
3 standard deviations (p=0.0027) throughout the trial. The final results may then be interpreted 
as having close to nominal significance levels. This rule appropriately requires very strong 
evidence for early stopping, is more conservative than the Pocock86 and O’Brien-Fleming87 rules 
and the alpha-spending function,88 and can be conducted at convenient times without inducing 
statistical complexity.89 
While these rules are intended for the primary endpoints, the goal of VITAL is to assess the 
overall balance of benefits and risks of the two agents in the primary prevention of cancer and 
CVD. Thus, consideration will also be given to the secondary endpoints that are needed in the 
interpretation of overall results. In addition, the monitoring rules will serve solely as guidelines in 
Table 3. Power for effects of a single agent on CVD in a factorial trial of 10,000 men 
aged ≥60 and 10,000 women aged ≥65, with 5 years of follow-up 
Observed 
RR† 
True 
RR 
Major 
CVD 
Total 
CVD 
CVD 
Mortality 
MI 
Stroke 
0.90 
0.875 
38.1 
57.3 
- 
- 
- 
0.85 
0.812 
71.1 
90.2 
- 
- 
- 
0.80 
0.750 
92.5 
99.2 
51.8 
53.5 
56.1 
0.75 
0.687 
99.0 
>99.9 
71.9 
73.8 
76.3 
0.70 
0.625 
99.9 
>99.9 
87.1 
88.5 
90.3 
0.65 
0.560 
>99.9 
>99.9 
95.6 
96.3 
97.1 
0.60 
0.500 
>99.9 
>99.9 
98.9 
99.1 
99.4 
†Observed RR=intent-to-treat RR, including noncompliant participants. Compliance is 
assumed to be 80%. True RR=that with perfect compliance. 

 
5
decisions regarding continuation or stopping of treatment arms. All decisions must be made 
after examining the totality of evidence, including other trial data, on these agents. 
 
 
X. REFERENCES 
 
1. 
Davis CD, Hartmuller V, Freedman DM, Hartge P, Picciano MF, Swanson CA, Milner JA. 
Vitamin D and cancer: current dilemmas and future needs. Nutr Rev 2007; 65:S71-4. 
2. 
Institute of Medicine Food and Nutrition Board. Dietary Reference Intakes: Calcium, 
Phosphorous, Magnesium, Vitamin D and Fluoride. Washington DC: National 
Academies Press, 1999. 
3. 
Giovannucci E. Strengths and limitations of current epidemiologic studies: vitamin D as a 
modifier of colon and prostate cancer risk. Nutr Rev 2007; 65:S77-9. 
4. 
Looker AC, Dawson-Hughes B, Calvo MS, Gunter EW, Sahyoun NR. Serum 25-
hydroxyvitamin D status of adolescents and adults in two seasonal subpopulations from 
NHANES III. Bone 2002; 30:771-7. 
5. 
Holick MF. Vitamin D deficiency. N Engl J Med 2007; 357:266-81. 
6. 
Harris SS. Vitamin D and African Americans. J Nutr 2006; 136:1126-9. 
7. 
Moore CE, Murphy MM, Holick MF. Vitamin D intakes by children and adults in the 
United States differ among ethnic groups. J Nutr 2005; 135:2478-85. 
8. 
Martins D, Wolf M, Pan D, Zadshir A, Tareen N, Thadhani R, Felsenfeld A, Levine B, 
Mehrotra R, Norris K. Prevalence of cardiovascular risk factors and the serum levels of 
25-hydroxyvitamin D in the United States: data from the Third National Health and 
Nutrition Examination Survey. Arch Intern Med 2007; 167:1159-65. 
9. 
Harris SS, Dawson-Hughes B. Reduced sun exposure does not explain the inverse 
association of 25-hydroxyvitamin D with percent body fat in older adults. J Clin 
Endocrinol Metab 2007; 92:3155-7. 
10. 
Hedley AA, Ogden CL, Johnson CL, Carroll MD, Curtin LR, Flegal KM. Prevalence of 
overweight and obesity among US children, adolescents, and adults, 1999-2002. JAMA 
2004; 291:2847-50. 
11. 
Ogden CL, Carroll MD, Curtin LR, McDowell MA, Tabak CJ, Flegal KM. Prevalence of 
overweight and obesity in the United States, 1999-2004. JAMA 2006; 295:1549-55. 
12. 
Kris-Etherton PM, Harris WS, Appel LJ. Fish consumption, fish oil, omega-3 fatty acids, 
and cardiovascular disease. Circulation 2002; 106:2747-57. 
13. 
NIH Office of Dietary Supplements, National Heart Lung and Blood Institute. Working 
Group Report on Future Clinical Research Directions on Omega-3 Fatty Acids and 
Cardiovascular Disease. June 2, 2004. Bethesda MD. Available at: 
http://www.nhlbi.nih.gov/meetings/workshops/omega-3/omega-3-rpt.htm Accessed on 
March 28, 2008. 
14. 
Stipp D. Fish-oil doses can be hard to swallow. Wall Street Journal: January 8, 2008; 
Page D1-D2. 
15. 
Gosnell M. Top 100 science stories of 2007: #8: Can vitamin D save your life? Discover: 
December 12, 2007. Available at http://discovermagazine.com/2008/jan/year-in-science-
2007/can-vitamin-d-save-your-life Accessed March 25, 2008. 
16. 
Guthrie C. The 10 biggest medical breakthroughs. #10: Benefits of vitamin D. Time: 
December 24, 2007. Available at: 
http://www.time.com/time/specials/2007/top10/article/0,30583,1686204_1686252_16903
93,00.html  Accessed March 25, 2008. 
17. 
Harvard Health Letter. The top 10 health stories of 2006. Harv Health Lett 2006; 32:1-3. 
18. 
Frisinger C. New year, new you: 8 simple rules for a healthier 2008. #1. Get more 
vitamin D. Fort Worth Star-Telegram (Texas): January 1, 2008; Page E1. 

 
6
19. 
Brody JE. An oldie vies for nutrient of the decade. New York Times: February 19, 2008; 
Page F7. 
20. 
Stampfer MJ. Vitamin D in the spotlight. Newsweek: December 11, 2006. Available at: 
http://www.newsweek.com/id/43998 Accessed March 25, 2008. 
21. 
Peng T. Don't forget your vitamins. Newsweek: March 24, 2008; Page 78. 
22. 
Rosenfeld I. We may need more vitamin D. Parade Magazine: January 29, 2006; Page 
15. 
23. 
Rhodes P. Vitamin D essentials: what you need to know about this important nutrient 
and where best to obtain it. Cooking Light 2008; 22:44-50. 
24. 
Foreman J. Is there any way to tell whether you are getting enough vitamin D? Boston 
Globe: February 4, 2008; Page C2.  
25. 
Harvard Health Letter. A more D-manding diet. Some experts say we should have a lot 
more vitamin D in our diets because it's protective against several diseases. Harv Health 
Lett 2007; 32:3.  
26. 
Simopoulos AP, Robinson J. The Omega Diet: The Lifesaving Nutritional Program 
Based on the Diet of the Island of Crete. New York: HarperCollins, 1999. 
27. 
Sears B. The Omega Rx Zone: The Miracle of the New High-Dose Fish Oil. New York: 
HarperCollins, 2004. 
28. 
Kaufman M. Fish oil may aid against manic depression. Washington Post, 1999:Z07. 
29. 
Foreman J. Fatty acid imbalance hurts our health. Boston Globe: February 10, 2004. 
30. 
Bakalar N. Fish oil linked to lower Alzheimer's risk. New York Times: November 14, 
2006. 
31. 
Munoz SS. Shopping around: DHA-enhanced foods. Wall Street Journal: March 27, 
2008; Page D2. 
32. 
Canadian Cancer Society. Canadian Cancer Society Announces Vitamin D 
Recommendation. Available at: 
http://www.cancer.ca/ccs/internet/mediareleaselist/0,,3172_1613121606_1997621989_l
angId-en.html. Accessed May 6, 2008. 
33. 
Vieth R, Bischoff-Ferrari H, Boucher BJ, Dawson-Hughes B, Garland CF, Heaney RP, 
Holick MF, Hollis BW, Lamberg-Allardt C, McGrath JJ, Norman AW, Scragg R, Whiting 
SJ, Willett WC, Zittermann A. The urgent need to recommend an intake of vitamin D that 
is effective. Am J Clin Nutr 2007; 85:649-50. 
34. 
Davis CD, Dwyer JT. The "sunshine vitamin": benefits beyond bone? J Natl Cancer Inst 
2007; 99:1563-5. 
35. 
Bardia A, Tleyjeh IM, Cerhan JR, Sood AK, Limburg PJ, Erwin PJ, Montori VM. Efficacy 
of antioxidant supplementation in reducing primary cancer incidence and mortality: 
systematic review and meta-analysis. Mayo Clin Proc 2008; 83:23-34. 
36. 
Mosca L, Banka CL, Benjamin EJ, Berra K, Bushnell C, Dolor RJ, Ganiats TG, Gomes 
AS, Gornik HL, Gracia C, Gulati M, Haan CK, Judelson DR, Keenan N, Kelepouris E, 
Michos ED, Newby LK, Oparil S, Ouyang P, Oz MC, Petitti D, Pinn VW, Redberg RF, 
Scott R, Sherif K, Smith SC, Jr., Sopko G, Steinhorn RH, Stone NJ, Taubert KA, Todd 
BA, Urbina E, Wenger NK. Evidence-based guidelines for cardiovascular disease 
prevention in women: 2007 update. Circulation 2007; 115:1481-501. 
37. 
Kris-Etherton PM, Lichtenstein AH, Howard BV, Steinberg D, Witztum JL. Antioxidant 
vitamin supplements and cardiovascular disease. Circulation 2004; 110:637-41. 
38. 
Lee IM, Cook NR, Gaziano JM, Gordon D, Ridker PM, Manson JE, Hennekens CH, 
Buring JE. Vitamin E in the primary prevention of cardiovascular disease and cancer: the 
Women's Health Study: a randomized controlled trial. JAMA 2005; 294:56-65. 
39. 
Miller ER, 3rd, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E. Meta-
analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann 
Intern Med 2005; 142:37-46. 

 
7
40. 
Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C. Mortality in randomized 
trials of antioxidant supplements for primary and secondary prevention: systematic 
review and meta-analysis. JAMA 2007; 297:842-57. 
41. 
Yokoyama M, Origasa H, Matsuzaki M, Matsuzawa Y, Saito Y, Ishikawa Y, Oikawa S, 
Sasaki J, Hishida H, Itakura H, Kita T, Kitabatake A, Nakaya N, Sakata T, Shimada K, 
Shirato K. Effects of eicosapentaenoic acid on major coronary events in 
hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint 
analysis. Lancet 2007; 369:1090-8. 
42. 
Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico. Dietary 
supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial 
infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della 
Sopravvivenza nell'Infarto miocardico. Lancet 1999; 354:447-55. 
43. 
Lappe JM, Travers-Gustafson D, Davies KM, Recker RR, Heaney RP. Vitamin D and 
calcium supplementation reduces cancer risk: results of a randomized trial. Am J Clin 
Nutr 2007; 85:1586-91. 
44. 
Wactawski-Wende J, Kotchen JM, Anderson GL, Assaf AR, Brunner RL, O'Sullivan MJ, 
Margolis KL, Ockene JK, Phillips L, Pottern L, Prentice RL, Robbins J, Rohan TE, Sarto 
GE, Sharma S, Stefanick ML, Van Horn L, Wallace RB, Whitlock E, Bassford T, 
Beresford SA, Black HR, Bonds DE, Brzyski RG, Caan B, Chlebowski RT, Cochrane B, 
Garland C, Gass M, Hays J, Heiss G, Hendrix SL, Howard BV, Hsia J, Hubbell FA, 
Jackson RD, Johnson KC, Judd H, Kooperberg CL, Kuller LH, LaCroix AZ, Lane DS, 
Langer RD, Lasser NL, Lewis CE, Limacher MC, Manson JE. Calcium plus vitamin D 
supplementation and the risk of colorectal cancer. N Engl J Med 2006; 354:684-96. 
45. 
Hathcock JN, Shao A, Vieth R, Heaney R. Risk assessment for vitamin D. Am J Clin 
Nutr 2007; 85:6-18. 
46. 
Lang JM, Buring JE, Rosner B, Cook N, Hennekens CH. Estimating the effect of the run-
in on the power of the Physicians' Health Study. Stat Med 1991; 10:1585-93. 
47. 
Talwar SA, Aloia JF, Pollack S, Yeh JK. Dose response to vitamin D supplementation 
among postmenopausal African American women. Am J Clin Nutr 2007; 86:1657-62. 
48. 
Prentice RL. A case-cohort design for epidemiologic cohort studies and disease 
prevention trials. Biometrika 1986; 73:1-11. 
49. 
Rimm EB, Giovannucci EL, Stampfer MJ, Colditz GA, Litin LB, Willett WC. 
Reproducibility and validity of an expanded self-administered semiquantitative food 
frequency questionnaire among male health professionals. Am J Epidemiol 1992; 
135:1114-26. 
50. 
Feskanich D, Rimm EB, Giovannucci EL, Colditz GA, Stampfer MJ, Litin LB, Willett WC. 
Reproducibility and validity of food intake measurements from a semiquantitative food 
frequency questionnaire. J Am Diet Assoc 1993; 93:790-6. 
51. 
Salvini S, Hunter DJ, Sampson L, Stampfer MJ, Colditz GA, Rosner B, Willett WC. Food-
based validation of a dietary questionnaire: the effects of week-to-week variation in food 
consumption. Int J Epidemiol 1989; 18:858-67. 
52. 
Jacques PF, Sulsky SI, Sadowski JA, Phillips JC, Rush D, Willett WC. Comparison of 
micronutrient intake measured by a dietary questionnaire and biochemical indicators of 
micronutrient status. Am J Clin Nutr 1993; 57:182-9. 
53. 
Feskanich D, Willett WC, Colditz GA. Calcium, vitamin D, milk consumption, and hip 
fractures: a prospective study among postmenopausal women. Am J Clin Nutr 2003; 
77:504-11. 
54. 
Hunter DJ, Rimm EB, Sacks FM, Stampfer MJ, Colditz GA, Litin LB, Willett WC. 
Comparison of measures of fatty acid intake by subcutaneous fat aspirate, food 
frequency questionnaire, and diet records in a free-living population of US men. Am J 
Epidemiol 1992; 135:418-27. 

 
8
55. 
Vieth R. Critique of the considerations for establishing the tolerable upper intake level for 
vitamin D: critical need for revision upwards. J Nutr 2006; 136:1117-22. 
56. 
Bischoff-Ferrari HA, Giovannucci E, Willett WC, Dietrich T, Dawson-Hughes B. 
Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health 
outcomes. Am J Clin Nutr 2006; 84:18-28. 
57. 
Dawson-Hughes B, Heaney RP, Holick MF, Lips P, Meunier PJ, Vieth R. Estimates of 
optimal vitamin D status. Osteoporos Int 2005; 16:713-6. 
58. 
Giovannucci E, Liu Y, Rimm EB, Hollis BW, Fuchs CS, Stampfer MJ, Willett WC. 
Prospective study of predictors of vitamin D status and cancer incidence and mortality in 
men. J Natl Cancer Inst 2006; 98:451-9. 
59. 
Aloia JF, Patel M, DiMaano R, Li-Ng M, Talwar SA, Mikhail M, Pollack S, Yeh  
            JK. Vitamin D intake to attain a desired serum 25-hydroxyvitamin D concentration. Am J 
Clin Nutr 2008;87:1952-1958 
60. 
Harris WS. International recommendations for consumption of long-chain omega-3 fatty 
acids. J Cardiovasc Med (Hagerstown) 2007; 8 Suppl 1:S50-2. 
61. 
Lee JH, O'Keefe JH, Lavie CJ, Marchioli R, Harris WS. Omega-3 fatty acids for 
cardioprotection. Mayo Clin Proc 2008; 83:324-32. [Docket No. 86G-0289], 1997. 
62. 
Agency for Healthcare Research and Quality. Effects of Omega-3 Fatty Acids on 
Cardiovascular Disease. Evidence Report/Technology Assessment Number 94. AHRQ 
Pub. No. 04-E009-1. 2004. Available at: 
http://www.ahrq.gov/clinic/epcsums/o3cardsum.htm Accessed March 28, 2008. 
63. 
Simopoulos AP. Evolutionary aspects of diet, the omega-6/omega-3 ratio and genetic 
variation: nutritional implications for chronic diseases. Biomed Pharmacother 2006; 
60:502-7. 
64. 
Kris-Etherton PM, Taylor DS, Yu-Poth S, Huth P, Moriarty K, Fishell V, Hargrove RL, 
Zhao G, Etherton TD. Polyunsaturated fatty acids in the food chain in the United States. 
Am J Clin Nutr 2000; 71:179S-88S. 
65. 
Harris WS. The omega-6/omega-3 ratio and cardiovascular disease risk: uses and 
abuses. Curr Atheroscler Rep 2006; 8:453-9. 
66. 
Stanley JC, Elsom RL, Calder PC, Griffin BA, Harris WS, Jebb SA, Lovegrove JA, Moore 
CS, Riemersma RA, Sanders TA. UK Food Standards Agency Workshop Report: the 
effects of the dietary n-6:n-3 fatty acid ratio on cardiovascular health. Br J Nutr 2007; 
98:1305-10. 
67. 
Harris WS, Poston WC, Haddock CK. Tissue n-3 and n-6 fatty acids and risk for 
coronary heart disease events. Atherosclerosis 2007; 193:1-10. 
68. 
Mozaffarian D, Ascherio A, Hu FB, Stampfer MJ, Willett WC, Siscovick DS, Rimm EB. 
Interplay between different polyunsaturated fatty acids and risk of coronary heart 
disease in men. Circulation 2005; 111:157-64. 
69. 
Department of Health and Human Services, U.S. Food and Drug Administration. 
Substances affirmed as generally recognized as safe: menhaden oil. Federal Register. 
June 5, 1997. Vol 62, No 108: pp 30751-30757. 21 CFR Part 184 [Docket No. 86G-
0289], 1997.52. 
Jacques PF, Sulsky SI, Sadowski JA, Phillips JC, Rush D, Willett 
WC. Comparison of micronutrient intake measured by a dietary questionnaire and 
biochemical indicators of micronutrient status. Am J Clin Nutr 1993; 57:182-9. 
70. 
Harris WS. Expert opinion: omega-3 fatty acids and bleeding--cause for concern? Am J 
Cardiol 2007; 99:44C-46C. 
71. 
Bays HE. Safety considerations with omega-3 fatty acid therapy. Am J Cardiol 2007; 
99:35C-43C. 
72. 
Satterfield S, Greco PJ, Goldhaber SZ, Stampfer MJ, Swartz SL, Stein EA, Kaplan L, 
Hennekens CH. Biochemical markers of compliance in the Physicians' Health Study. Am 
J Prev Med 1990; 6:290-4. 

 
9
73. 
Fritz AG, Percy C, Jack A, Shanmugaratham K, Sobin LH, Parkin MD, Whelan S. 
International Classification of Diseases for Oncology (ICD-O), Third Edition. Geneva: 
World Health Organization, 2000. 
74. 
Thygesen K, Alpert JS, White HD, Joint ESC/ACCF/AHA/WHF Task Force for the 
Redefinition of Myocardial Infarction. Universal definition of myocardial infarction. 
Circulation 2007; 116:2634-53. 
75. 
Adams HP, Jr., Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, Marsh EE, 3rd. 
Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter 
clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke 1993; 24:35-
41. 
76. 
Cox DR. Regression models and life-tables. J Royal Statist Soc B 1972; 34:187-220. 
77. 
Gooley TA, Leisenring W, Crowley J, Storer BE. Estimation of failure probabilities in the 
presence of competing risks: new representations of old estimators. Stat Med 1999; 
18:695-706. 
78. 
Putter H, Fiocco M, Geskus RB. Tutorial in biostatistics: competing risks and multi-state 
models. Stat Med 2007; 26:2389-430. 
79. 
Fine JP, Gray RJ. A proportional hazards model for the subdistribution of a competing 
risk. J Am Stat Assoc 1999; 94:496-509. 
80. 
Barlow WE. Robust variance estimation for the case-cohort design. Biometrics 1994; 
50:1064-72. 
81. 
Borgan O, Langholz B, Samuelsen SO, Goldstein L, Pogoda J. Exposure stratified case-
cohort designs. Lifetime Data Anal 2000; 6:39-58. 
82. 
Holick MF. Vitamin D for health and in chronic kidney disease. Semin Dial 2005; 18:266-
75 
83. 
Freedman LS. Tables of the number of patients required in clinical trials using the 
logrank test. Stat Med 1982; 1:121-9. 
84. 
Haybittle JL. Repeated assessment of results in clinical trials of cancer treatment. Br J 
Radiol 1971; 44:793-7. 
85. 
Peto R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard SV, Mantel N, McPherson 
K, Peto J, Smith PG. Design and analysis of randomized clinical trials requiring 
prolonged observation of each patient. I. Introduction and design. Br J Cancer 1976; 
34:585-612. 
86. 
Geller NL, Pocock SJ. Interim analyses in randomized clinical trials: ramifications and 
guidelines for practitioners. Biometrics 1987; 43:213-23. 
87. 
O'Brien PC, Fleming TR. A multiple testing procedure for clinical trials. Biometrics 1979; 
35:549-56. 
88. 
DeMets DL, Lan KK. Interim analysis: the alpha spending function approach. Stat Med 
1994; 13:1341-52. 
89. 
Pocock SJ. Current controversies in data monitoring for clinical trials. Clin Trials 2006; 
3:513-21. 
 

 
1
 PROTOCOL FOR THE VITAL TRIAL  
November 30, 2017, version 23 
 
 
VI. 
BIOSTATISTICAL ANALYSIS 
A. DATA ANALYSIS  
Analyses of treatment effects will be based on the intent-to-treat principle. The first analysis will 
compare baseline characteristics by randomized treatment assignment to ensure that balance 
was achieved by the randomization. Characteristics to be examined include known risk factors 
for cancer and CVD, including age; gender; race/ethnicity; BMI; smoking; alcohol use; physical 
activity; medical conditions such as hypertension, hyperlipidemia, diabetes, and family history of 
cancer and CVD; and baseline vitamin D and omega-3 fatty acid levels as assessed by dietary 
questionnaire in all participants and blood assays in a case-cohort subsample. The large 
sample size, as well as successful balance of known potential confounders, will provide 
assurance that unmeasured or unknown potential confounders will also be equally distributed 
across randomized treatment groups. 
In this 2x2 factorial design, the primary aim is to compare the main effects of intention-to-treat 
with vitamin D softgel capsules and with Omacor® fish oil on cancer and CVD. We will use the 
Cox proportional hazards model to allow for variable follow-up lengths73 and will estimate the 
hazard ratio for each intervention using indicators for treatment exposure, controlling for the 
second intervention, age, and gender. Because the cohort will consist of older individuals, 
competing risks due to deaths from other causes will be considered. The primary analysis will 
estimate the cause-specific hazard and the hazard ratio comparing intervention groups for each 
outcome of interest by censoring individuals with deaths due to competing causes. To estimate 
the cumulative incidence function, the subdistribution of each endpoint will be plotted over 
time.74,75 The alternative Fine and Gray approach76 models the effect of treatment on the 
subdistribution hazard or directly on the cumulative incidence function. While we will consider 
this and compare its fit to the model-free cumulative incidence curves,75 the proportional 
hazards approach will be our primary analysis. 
Similar analyses will be conducted for both cancer and CVD. For cancer, we will estimate the 
intervention effects on the primary endpoint of total cancer, as well as the secondary outcomes 
of breast, prostate, and colorectal cancer, and cancer mortality. For CVD we will estimate the 
effects on the primary composite endpoint of major CVD (MI, stroke, and cardiovascular 
mortality); on the secondary composite endpoint that additionally includes coronary 
revascularization; and on the individual components of MI, stroke, revascularization, and 
cardiovascular mortality. 
Beyond the primary analyses, we will conduct prespecified secondary analyses to examine 
effect modification by the other randomized intervention, by baseline risk factors, by compliance, 
and by time. We have a particular interest in exploring interactions between the interventions, as 
well as with the baseline plasma biomarkers of 25(OH)D for vitamin D and of EPA+DHA for fish 
oil. We hypothesize that the intervention effects may be larger among those with below-median 
baseline levels, and will examine treatment effects by quartiles of these biomarkers. The 
biomarker data will be analyzed as a case-cohort study using methods for proportional hazards 
regression.77 Because of the frequency matching by age and gender, appropriate stratum-
specific weighting of the observations will be employed.78  We also have a prior interest in the 
effects of the vitamin D intervention within groups defined by race/ethnicity and skin 

 
2
pigmentation, and by BMI. In addition, we will evaluate effect modification by age and gender, 
as well as by sunlight exposure, calcium and phosphorus intakes from the FFQ (as these 
nutrients affect vitamin D bioavailability79), and baseline risk factors for cancer and CVD. The 
latter interaction effects will be interpreted cautiously, as hypothesis generating. We will also 
examine as a prespecified secondary analysis the association among compliers, in which 
participants’ follow-up time will be censored when they are no longer compliant with taking at 
least two-thirds of their assigned active agents or placebos. Finally, we will examine whether 
treatment effects vary over time and duration of treatment by examining survival plots and 
interactions with time.  We will additionally conduct analyses for the primary endpoints of cancer 
and cardiovascular disease excluding events occurring in Year 1, as well as in Years 1 and 2, of 
follow-up. There may be a latent effect on both outcomes, depending on the mechanisms by 
which these agents act. 
We will also compare the incidence of potential side effects in the active v. placebo groups for 
each agent, including the incidence of kidney stones with vitamin D assignment (cf. WHI 
calcium-vitamin D findings44) and the incidence of GI symptoms, skin abnormalities, and 
bleeding with fish oil assignment (cf. JELIS findings41). 
B. POWER CALCULATIONS  
To calculate power, the following assumptions were made: (1) a 2x2 factorial trial in 12,500 men 
aged >50 and 12,500 women aged >55; (2) independent and equal allocation of participants to 
each treatment; (3) an age distribution based on that observed at baseline in our past trials for 
men aged ≥50 and women aged ≥55; (4) age-specific event rates based on the observed rates 
in the first 5 years of the PHS II for men and the WHS for women; (5) trial follow-up of 5 years, 
with little loss to follow-up as achieved in our past trials; and (6) compliance similar to that in 
published trials upon which our estimated rate ratio (RR) reductions are based. The cited 
reductions are thus the observed effects we would see in the trial. The corresponding ‘true’ RR 
is given assuming an average compliance of 80%. Power is given for a two-sided test using a 
logrank analysis80 with a significance level of 0.05. 
Cancer: Assuming that only one agent is effective, with 5 years of treatment and follow-up we 
will have 93% power to detect an observed RR of 0.85 for the primary endpoint of total cancer 
incidence (Table 1). For 
prostate cancer, we will 
have 93% power to 
detect a 25% risk 
reduction. For breast 
cancer, we will have 
86% power to detect a 
35% risk reduction. For 
colorectal cancer, we 
will have 77% power to 
detect a 35% risk 
reduction and 88% 
power to detect a 40% 
risk reduction. For 
cancer mortality, we will 
have 86% power to detect a 30% risk reduction. If both agents are effective in preventing cancer 
but act independently, power would be reduced slightly due to a somewhat smaller overall 

 
3
number of events. If the agents interact, however, power will be affected to the extent of the 
interaction. Should the agents act synergistically, power would increase, as illustrated in Table 2 
for the outcome of total cancer.  
For example, if the effect of each agent alone is a reduction of 10%, but in combination the 
effect is stronger, with an additional 10% decrease, the RR comparing the combined group to 
the all placebo group would be 0.73, versus 0.81 with additive effects (on the multiplicative 
scale). Power for the main effect of each agent would then increase to 88% (or higher with 
greater synergy). The trial would have power to detect moderate-to-large interactions, as shown 
in Table 2. Should the agents interact in a subadditive fashion, power would be reduced to the 
extent of the subadditivity. Power to explore effect modification by baseline blood levels of 
25(OH)D and EPA+DHA 
among those above and 
below the median level 
in the cohort will be 
lower because we 
anticipate that about 
60% of participants will 
provide blood 
specimens.  
Power has been shown 
for a given observed 
reduction in risk, 
assuming compliance 
similar to that seen in current randomized trials such as the WHS and PHS. For total cancer, our 
power will be excellent with a 5-year follow-up even if compliance is lower. 
CVD: As shown in Table 3, 
assuming that only one 
agent is effective, with 5 
years of treatment and 
follow-up we will have 95% 
power to detect an observed 
RR of 0.80 for the primary 
composite endpoint of MI, 
stroke, and cardiovascular 
mortality. For the secondary 
composite endpoint of MI, 
stroke, cardiovascular 
mortality, and coronary 
revascularization, we will 
have over 99% power to 
detect a 20% reduction and 
93% power to detect a 15% 
reduction. With a RR of 0.70 
we will have 89% power for 
cardiovascular mortality, 
and we will have over 80% power to detect an RR of 0.75 for MI and stroke. If both agents are 
effective in preventing CVD but act independently, power would be reduced only slightly due to 

 
4
a smaller overall number of events. If the agents act synergistically, power would increase, as 
described for cancer. If the agents interact in subadditive fashion, power would be reduced to 
the extent of the subadditivity.  
 
X. REFERENCES 
 
1. 
Davis CD, Hartmuller V, Freedman DM, Hartge P, Picciano MF, Swanson CA, Milner JA. 
Vitamin D and cancer: current dilemmas and future needs. Nutr Rev 2007; 65:S71-4. 
2. 
Institute of Medicine. Dietary Reference Intakes for Calcium and Vitamin D. Washington 
DC: National Academies Press, 2011. 
3. 
Giovannucci E. Strengths and limitations of current epidemiologic studies: vitamin D as a 
modifier of colon and prostate cancer risk. Nutr Rev 2007; 65:S77-9. 
4. 
Looker AC, Dawson-Hughes B, Calvo MS, Gunter EW, Sahyoun NR. Serum 25-
hydroxyvitamin D status of adolescents and adults in two seasonal subpopulations from 
NHANES III. Bone 2002; 30:771-7. 
5. 
Holick MF. Vitamin D deficiency. N Engl J Med 2007; 357:266-81. 
6. 
Harris SS. Vitamin D and African Americans. J Nutr 2006; 136:1126-9. 
7. 
Moore CE, Murphy MM, Holick MF. Vitamin D intakes by children and adults in the 
United States differ among ethnic groups. J Nutr 2005; 135:2478-85. 
8. 
Martins D, Wolf M, Pan D, Zadshir A, Tareen N, Thadhani R, Felsenfeld A, Levine B, 
Mehrotra R, Norris K. Prevalence of cardiovascular risk factors and the serum levels of 
25-hydroxyvitamin D in the United States: data from the Third National Health and 
Nutrition Examination Survey. Arch Intern Med 2007; 167:1159-65. 
9. 
Harris SS, Dawson-Hughes B. Reduced sun exposure does not explain the inverse 
association of 25-hydroxyvitamin D with percent body fat in older adults. J Clin 
Endocrinol Metab 2007; 92:3155-7. 
10. 
Hedley AA, Ogden CL, Johnson CL, Carroll MD, Curtin LR, Flegal KM. Prevalence of 
overweight and obesity among US children, adolescents, and adults, 1999-2002. JAMA 
2004; 291:2847-50. 
11. 
Ogden CL, Carroll MD, Curtin LR, McDowell MA, Tabak CJ, Flegal KM. Prevalence of 
overweight and obesity in the United States, 1999-2004. JAMA 2006; 295:1549-55. 
12. 
Kris-Etherton PM, Harris WS, Appel LJ. Fish consumption, fish oil, omega-3 fatty acids, 
and cardiovascular disease. Circulation 2002; 106:2747-57. 
13. 
NIH Office of Dietary Supplements, National Heart Lung and Blood Institute. Working 
Group Report on Future Clinical Research Directions on Omega-3 Fatty Acids and 
Cardiovascular Disease. June 2, 2004. Bethesda MD. Available at: 
http://www.nhlbi.nih.gov/meetings/workshops/omega-3/omega-3-rpt.htm Accessed on 
March 28, 2008. 
14. 
Stipp D. Fish-oil doses can be hard to swallow. Wall Street Journal: January 8, 2008; 
Page D1-D2. 
15. 
Gosnell M. Top 100 science stories of 2007: #8: Can vitamin D save your life? Discover: 
December 12, 2007. Available at http://discovermagazine.com/2008/jan/year-in-science-
2007/can-vitamin-d-save-your-life Accessed March 25, 2008. 
16. 
Guthrie C. The 10 biggest medical breakthroughs. #10: Benefits of vitamin D. Time: 
December 24, 2007. Available at: 
http://www.time.com/time/specials/2007/top10/article/0,30583,1686204_1686252_16903
93,00.html  Accessed March 25, 2008. 
17. 
Harvard Health Letter. The top 10 health stories of 2006. Harv Health Lett 2006; 32:1-3. 

 
5
18. 
Frisinger C. New year, new you: 8 simple rules for a healthier 2008. #1. Get more 
vitamin D. Fort Worth Star-Telegram (Texas): January 1, 2008; Page E1. 
19. 
Brody JE. An oldie vies for nutrient of the decade. New York Times: February 19, 2008; 
Page F7. 
20. 
Stampfer MJ. Vitamin D in the spotlight. Newsweek: December 11, 2006. Available at: 
http://www.newsweek.com/id/43998 Accessed March 25, 2008. 
21. 
Peng T. Don't forget your vitamins. Newsweek: March 24, 2008; Page 78. 
22. 
Rosenfeld I. We may need more vitamin D. Parade Magazine: January 29, 2006; Page 
15. 
23. 
Rhodes P. Vitamin D essentials: what you need to know about this important nutrient 
and where best to obtain it. Cooking Light 2008; 22:44-50. 
24. 
Foreman J. Is there any way to tell whether you are getting enough vitamin D? Boston 
Globe: February 4, 2008; Page C2.  
25. 
Harvard Health Letter. A more D-manding diet. Some experts say we should have a lot 
more vitamin D in our diets because it's protective against several diseases. Harv Health 
Lett 2007; 32:3.  
26. 
Simopoulos AP, Robinson J. The Omega Diet: The Lifesaving Nutritional Program 
Based on the Diet of the Island of Crete. New York: HarperCollins, 1999. 
27. 
Sears B. The Omega Rx Zone: The Miracle of the New High-Dose Fish Oil. New York: 
HarperCollins, 2004. 
28. 
Kaufman M. Fish oil may aid against manic depression. Washington Post, 1999:Z07. 
29. 
Foreman J. Fatty acid imbalance hurts our health. Boston Globe: February 10, 2004. 
30. 
Bakalar N. Fish oil linked to lower Alzheimer's risk. New York Times: November 14, 
2006. 
31. 
Munoz SS. Shopping around: DHA-enhanced foods. Wall Street Journal: March 27, 
2008; Page D2. 
32. 
Canadian Cancer Society. Canadian Cancer Society Announces Vitamin D 
Recommendation. Available at: 
http://www.cancer.ca/ccs/internet/mediareleaselist/0,,3172_1613121606_1997621989_l
angId-en.html. Accessed May 6, 2008. 
33. 
Vieth R, Bischoff-Ferrari H, Boucher BJ, Dawson-Hughes B, Garland CF, Heaney RP, 
Holick MF, Hollis BW, Lamberg-Allardt C, McGrath JJ, Norman AW, Scragg R, Whiting 
SJ, Willett WC, Zittermann A. The urgent need to recommend an intake of vitamin D that 
is effective. Am J Clin Nutr 2007; 85:649-50. 
34. 
Davis CD, Dwyer JT. The "sunshine vitamin": benefits beyond bone? J Natl Cancer Inst 
2007; 99:1563-5. 
35. 
Bardia A, Tleyjeh IM, Cerhan JR, Sood AK, Limburg PJ, Erwin PJ, Montori VM. Efficacy 
of antioxidant supplementation in reducing primary cancer incidence and mortality: 
systematic review and meta-analysis. Mayo Clin Proc 2008; 83:23-34. 
36. 
Mosca L, Banka CL, Benjamin EJ, Berra K, Bushnell C, Dolor RJ, Ganiats TG, Gomes 
AS, Gornik HL, Gracia C, Gulati M, Haan CK, Judelson DR, Keenan N, Kelepouris E, 
Michos ED, Newby LK, Oparil S, Ouyang P, Oz MC, Petitti D, Pinn VW, Redberg RF, 
Scott R, Sherif K, Smith SC, Jr., Sopko G, Steinhorn RH, Stone NJ, Taubert KA, Todd 
BA, Urbina E, Wenger NK. Evidence-based guidelines for cardiovascular disease 
prevention in women: 2007 update. Circulation 2007; 115:1481-501. 
37. 
Kris-Etherton PM, Lichtenstein AH, Howard BV, Steinberg D, Witztum JL. Antioxidant 
vitamin supplements and cardiovascular disease. Circulation 2004; 110:637-41. 
38. 
Lee IM, Cook NR, Gaziano JM, Gordon D, Ridker PM, Manson JE, Hennekens CH, 
Buring JE. Vitamin E in the primary prevention of cardiovascular disease and cancer: the 
Women's Health Study: a randomized controlled trial. JAMA 2005; 294:56-65. 

 
6
39. 
Miller ER, 3rd, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E. Meta-
analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann 
Intern Med 2005; 142:37-46. 
40. 
Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C. Mortality in randomized 
trials of antioxidant supplements for primary and secondary prevention: systematic 
review and meta-analysis. JAMA 2007; 297:842-57. 
41. 
Yokoyama M, Origasa H, Matsuzaki M, Matsuzawa Y, Saito Y, Ishikawa Y, Oikawa S, 
Sasaki J, Hishida H, Itakura H, Kita T, Kitabatake A, Nakaya N, Sakata T, Shimada K, 
Shirato K. Effects of eicosapentaenoic acid on major coronary events in 
hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint 
analysis. Lancet 2007; 369:1090-8. 
42. 
Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico. Dietary 
supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial 
infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della 
Sopravvivenza nell'Infarto miocardico. Lancet 1999; 354:447-55. 
43. 
Lappe JM, Travers-Gustafson D, Davies KM, Recker RR, Heaney RP. Vitamin D and 
calcium supplementation reduces cancer risk: results of a randomized trial. Am J Clin 
Nutr 2007; 85:1586-91. 
44. 
Wactawski-Wende J, Kotchen JM, Anderson GL, Assaf AR, Brunner RL, O'Sullivan MJ, 
Margolis KL, Ockene JK, Phillips L, Pottern L, Prentice RL, Robbins J, Rohan TE, Sarto 
GE, Sharma S, Stefanick ML, Van Horn L, Wallace RB, Whitlock E, Bassford T, 
Beresford SA, Black HR, Bonds DE, Brzyski RG, Caan B, Chlebowski RT, Cochrane B, 
Garland C, Gass M, Hays J, Heiss G, Hendrix SL, Howard BV, Hsia J, Hubbell FA, 
Jackson RD, Johnson KC, Judd H, Kooperberg CL, Kuller LH, LaCroix AZ, Lane DS, 
Langer RD, Lasser NL, Lewis CE, Limacher MC, Manson JE. Calcium plus vitamin D 
supplementation and the risk of colorectal cancer. N Engl J Med 2006; 354:684-96. 
45. 
Hathcock JN, Shao A, Vieth R, Heaney R. Risk assessment for vitamin D. Am J Clin 
Nutr 2007; 85:6-18. 
46. 
Lang JM, Buring JE, Rosner B, Cook N, Hennekens CH. Estimating the effect of the run-
in on the power of the Physicians' Health Study. Stat Med 1991; 10:1585-93. 
47. 
Talwar SA, Aloia JF, Pollack S, Yeh JK. Dose response to vitamin D supplementation 
among postmenopausal African American women. Am J Clin Nutr 2007; 86:1657-62. 
48. 
Rimm EB, Giovannucci EL, Stampfer MJ, Colditz GA, Litin LB, Willett WC. 
Reproducibility and validity of an expanded self-administered semiquantitative food 
frequency questionnaire among male health professionals. Am J Epidemiol 1992; 
135:1114-26. 
49. 
Feskanich D, Rimm EB, Giovannucci EL, Colditz GA, Stampfer MJ, Litin LB, Willett WC. 
Reproducibility and validity of food intake measurements from a semiquantitative food 
frequency questionnaire. J Am Diet Assoc 1993; 93:790-6. 
50. 
Salvini S, Hunter DJ, Sampson L, Stampfer MJ, Colditz GA, Rosner B, Willett WC. Food-
based validation of a dietary questionnaire: the effects of week-to-week variation in food 
consumption. Int J Epidemiol 1989; 18:858-67. 
51. 
Jacques PF, Sulsky SI, Sadowski JA, Phillips JC, Rush D, Willett WC. Comparison of 
micronutrient intake measured by a dietary questionnaire and biochemical indicators of 
micronutrient status. Am J Clin Nutr 1993; 57:182-9. 
52. 
Feskanich D, Willett WC, Colditz GA. Calcium, vitamin D, milk consumption, and hip 
fractures: a prospective study among postmenopausal women. Am J Clin Nutr 2003; 
77:504-11. 
53. 
Hunter DJ, Rimm EB, Sacks FM, Stampfer MJ, Colditz GA, Litin LB, Willett WC. 
Comparison of measures of fatty acid intake by subcutaneous fat aspirate, food 

 
7
frequency questionnaire, and diet records in a free-living population of US men. Am J 
Epidemiol 1992; 135:418-27. 
54. 
Bischoff-Ferrari HA, Giovannucci E, Willett WC, Dietrich T, Dawson-Hughes B. 
Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health 
outcomes. Am J Clin Nutr 2006; 84:18-28. 
55. 
Dawson-Hughes B, Heaney RP, Holick MF, Lips P, Meunier PJ, Vieth R. Estimates of 
optimal vitamin D status. Osteoporos Int 2005; 16:713-6. 
56. 
Giovannucci E, Liu Y, Rimm EB, Hollis BW, Fuchs CS, Stampfer MJ, Willett WC. 
Prospective study of predictors of vitamin D status and cancer incidence and mortality in 
men. J Natl Cancer Inst 2006; 98:451-9. 
57. 
Aloia JF, Patel M, DiMaano R, Li-Ng M, Talwar SA, Mikhail M, Pollack S, Yeh  
            JK. Vitamin D intake to attain a desired serum 25-hydroxyvitamin D concentration. Am J 
Clin Nutr 2008;87:1952-1958 
58. 
Harris WS. International recommendations for consumption of long-chain omega-3 fatty 
acids. J Cardiovasc Med (Hagerstown) 2007; 8 Suppl 1:S50-2. 
59. 
Lee JH, O'Keefe JH, Lavie CJ, Marchioli R, Harris WS. Omega-3 fatty acids for 
cardioprotection. Mayo Clin Proc 2008; 83:324-32. [Docket No. 86G-0289], 1997. 
60. 
Agency for Healthcare Research and Quality. Effects of Omega-3 Fatty Acids on 
Cardiovascular Disease. Evidence Report/Technology Assessment Number 94. AHRQ 
Pub. No. 04-E009-1. 2004. Available at: 
http://www.ahrq.gov/clinic/epcsums/o3cardsum.htm Accessed March 28, 2008. 
61. 
Simopoulos AP. Evolutionary aspects of diet, the omega-6/omega-3 ratio and genetic 
variation: nutritional implications for chronic diseases. Biomed Pharmacother 2006; 
60:502-7. 
62. 
Kris-Etherton PM, Taylor DS, Yu-Poth S, Huth P, Moriarty K, Fishell V, Hargrove RL, 
Zhao G, Etherton TD. Polyunsaturated fatty acids in the food chain in the United States. 
Am J Clin Nutr 2000; 71:179S-88S. 
63. 
Harris WS. The omega-6/omega-3 ratio and cardiovascular disease risk: uses and 
abuses. Curr Atheroscler Rep 2006; 8:453-9. 
64. 
Stanley JC, Elsom RL, Calder PC, Griffin BA, Harris WS, Jebb SA, Lovegrove JA, Moore 
CS, Riemersma RA, Sanders TA. UK Food Standards Agency Workshop Report: the 
effects of the dietary n-6:n-3 fatty acid ratio on cardiovascular health. Br J Nutr 2007; 
98:1305-10. 
65. 
Harris WS, Poston WC, Haddock CK. Tissue n-3 and n-6 fatty acids and risk for 
coronary heart disease events. Atherosclerosis 2007; 193:1-10. 
66. 
Mozaffarian D, Ascherio A, Hu FB, Stampfer MJ, Willett WC, Siscovick DS, Rimm EB. 
Interplay between different polyunsaturated fatty acids and risk of coronary heart 
disease in men. Circulation 2005; 111:157-64. 
67. 
Department of Health and Human Services, U.S. Food and Drug Administration. 
Substances affirmed as generally recognized as safe: menhaden oil. Federal Register. 
June 5, 1997. Vol 62, No 108: pp 30751-30757. 21 CFR Part 184 [Docket No. 86G-
0289], 1997. 
68. 
Bays HE. Safety considerations with omega-3 fatty acid therapy. Am J Cardiol 2007; 
99:35C-43C. 
69. 
Satterfield S, Greco PJ, Goldhaber SZ, Stampfer MJ, Swartz SL, Stein EA, Kaplan L, 
Hennekens CH. Biochemical markers of compliance in the Physicians' Health Study. Am 
J Prev Med 1990; 6:290-4. 
70. 
Fritz AG, Percy C, Jack A, Shanmugaratham K, Sobin LH, Parkin MD, Whelan S. 
International Classification of Diseases for Oncology (ICD-O), Third Edition. Geneva: 
World Health Organization, 2000. 

 
8
71. 
Thygesen K, Alpert JS, White HD, Joint ESC/ACCF/AHA/WHF Task Force for the 
Redefinition of Myocardial Infarction. Universal definition of myocardial infarction. 
Circulation 2007; 116:2634-53. 
72. 
Adams HP, Jr., Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, Marsh EE, 3rd. 
Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter 
clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke 1993; 24:35-
41. 
73. 
Cox DR. Regression models and life-tables. J Royal Statist Soc B 1972; 34:187-220. 
74. 
Gooley TA, Leisenring W, Crowley J, Storer BE. Estimation of failure probabilities in the 
presence of competing risks: new representations of old estimators. Stat Med 1999; 
18:695-706. 
75. 
Putter H, Fiocco M, Geskus RB. Tutorial in biostatistics: competing risks and multi-state 
models. Stat Med 2007; 26:2389-430. 
76. 
Fine JP, Gray RJ. A proportional hazards model for the subdistribution of a competing 
risk. J Am Stat Assoc 1999; 94:496-509. 
77. 
Barlow WE. Robust variance estimation for the case-cohort design. Biometrics 1994; 
50:1064-72. 
78. 
Borgan O, Langholz B, Samuelsen SO, Goldstein L, Pogoda J. Exposure stratified case-
cohort designs. Lifetime Data Anal 2000; 6:39-58. 
79. 
Holick MF. Vitamin D for health and in chronic kidney disease. Semin Dial 2005; 18:266-
75 
80. 
Freedman LS. Tables of the number of patients required in clinical trials using the 
logrank test. Stat Med 1982; 1:121-9. 
81. 
Brasky TM, Darke AK, Song X, et al. Plasma phospholipid fatty acids and prostate 
cancer risk in the SELECT trial. J Natl Cancer Inst 2013; 105:1132-41. 
82. 
Haybittle JL. Repeated assessment of results in clinical trials of cancer treatment. Br J 
Radiol 1971; 44:793-7. 
83. 
Peto R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard SV, Mantel N, McPherson 
K, Peto J, Smith PG. Design and analysis of randomized clinical trials requiring 
prolonged observation of each patient. I. Introduction and design. Br J Cancer 1976; 
34:585-612. 
84. 
Geller NL, Pocock SJ. Interim analyses in randomized clinical trials: ramifications and 
guidelines for practitioners. Biometrics 1987; 43:213-23. 
85. 
O'Brien PC, Fleming TR. A multiple testing procedure for clinical trials. Biometrics 1979; 
35:549-56. 
86. 
DeMets DL, Lan KK. Interim analysis: the alpha spending function approach. Stat Med 
1994; 13:1341-52. 
87. 
Pocock SJ. Current controversies in data monitoring for clinical trials. Clin Trials 2006; 
3:513-21. 
 

September 2018 
 
The VITamin D and OmegA-3 TriaL (VITAL): Summary of Changes to the Statistical Analysis Plan:   
 
January 28, 2010 (v. 2) was the date that the first full protocol was approved by the IRB. A copy of the 
data analysis plan in this protocol is provided. (Our initial IRB application, submitted in June 2009, 
contained a draft protocol.) 
 
June 8, 2011 (v.5) 
• 
Eligible age range was lowered to 50 years or older for males and 55 or older for females, to 
reflect the younger age of development of heart disease and cancer among underrepresented 
minorities, and thus facilitate minority recruitment. 
 
December 1, 2011 (v. 6) 
• 
Revised power calculations based on lowering the eligibility age range on 6/8/11.   
 
December 3, 2012 (v. 8) 
• 
Provided new power calculations based upon a study population of 25,000 
 
September 17, 2013 (v. 11) 
• 
Specified that the projected randomized population would be a maximum of 26,500 
 
September 13, 2017 (v. 22) 
• 
Specified that, at the conclusion of the randomized trial, we will send participants two additional 
annual questionnaires to collect supplemental study data during the post-intervention phase. 
 
November 30, 2017 (v. 23) is the current protocol.  A copy of the data analysis plan in this version is 
provided. 
• 
Clarified that we will additionally conduct analyses excluding events occurring in year 1 and 
years 1-2 of follow-up (our original protocol on 1/28/10 [v.2] specified: “Finally, we will examine 
whether treatment effects vary over time and duration of treatment by examining survival plots 
and interactions with time. There may be a latent effect on cancer incidence, depending on the 
stage of carcinogenesis during which these agents act.”  In the 11/30/17 protocol, we formalized 
this as analyses excluding events during year 1 and years 1-2 of follow-up.) 
 
• 
Data analysis section amplified:  specified sensitivity analyses by compliance, with participants’ 
follow-up time censored when they are no longer compliant with taking at least two-thirds of 
their assigned active agents or placebos (as in our previous trials). 
 
 
NOTE:  VITAL investigators were unblinded to randomization treatment codes on December 6, 2017 
prior to a teleconference with VITAL DSMB members.  All of the above protocol changes were made 
PRIOR to the investigator unblinding on December 6, 2017. The intervention phase of VITAL ended on 
December 31, 2017.