Evidence from a prespecified ancillary analysis of leukocyte telomere length within a large randomized controlled trial.
Study classification and identity
Study type
Randomized controlled trial, prespecified ancillary analysis
Parent study
VITamin D and OmegA-3 TriaL (VITAL)
Ancillary study focus
Longitudinal leukocyte telomere length dynamics
Exposure type
Randomized exposure to vitamin D₃ and/or marine omega-3 fatty acids
Intervention arms
Vitamin D₃ (2,000 IU/day)
Marine omega-3 fatty acids (1 g/day)
Combined vitamin D₃ and omega-3 fatty acids
Placebo
Primary endpoint category
Biological marker (leukocyte telomere length)
Clinical endpoints assessed
Not applicable in this study
Study purpose and framing
This prespecified ancillary study examined whether randomized exposure to vitamin D₃ and/or marine omega-3 fatty acids altered the longitudinal trajectory of leukocyte telomere length over four years within a large, double-blind, placebo-controlled randomized controlled trial.
Leukocyte telomere length was evaluated as a population-level biological marker reflecting cumulative cellular maintenance processes over time rather than as a clinical outcome or a direct measure of ageing phenotype.
Why leukocyte telomere length is used as a biological marker
Telomeres are repetitive nucleotide sequences located at the ends of chromosomes that preserve genomic stability during cell division. With successive replication cycles, telomeres progressively shorten, reflecting cumulative cellular replication history, oxidative stress exposure, and efficiency of telomere maintenance systems.
Because telomere shortening unfolds gradually, leukocyte telomere length is widely used in population research as a marker associated with long-term cellular ageing processes rather than acute physiological change. Baseline telomere length varies substantially across individuals, and longitudinal changes are interpreted at the group level.
Leukocyte telomere measurements represent an aggregate signal across circulating immune cell populations. Shifts in immune cell subtype composition over time can influence measured telomere length independently of telomere dynamics within specific cell types.
Study overview
The VITAL trial was a large, randomized, double-blind, placebo-controlled clinical trial conducted in the United States using a 2 × 2 factorial design. The parent trial enrolled 25,871 participants, including women aged 55 years and older and men aged 50 years and older, with follow-up of up to five years.
Participants were randomly assigned to receive vitamin D₃, marine omega-3 fatty acids, both interventions, or placebo. The telomere analysis was conducted as a prespecified ancillary study embedded within this trial framework.
Study design and measurement approach
The telomere ancillary study included 1,054 participants who attended in-person study visits at the Harvard Clinical and Translational Science Center. Leukocyte telomere length was measured at baseline, year 2, and year 4.
Telomere length was assessed using a quantitative polymerase chain reaction–based assay described as Absolute Human Telomere Length Quantification. This approach is suitable for longitudinal population studies but is subject to technical and biological variability and does not yield precise individual-level telomere measurements.
Outcome hierarchy
Directly measured
Change in leukocyte telomere length over time
Not measured
Clinical outcomes
Disease incidence
Functional ageing metrics
Lifespan or mortality
Not inferable from this study
Individual ageing rate
Disease risk modification
Causal biological mechanisms linking vitamin D₃ exposure to telomere maintenance
Statistical analysis
The prespecified primary outcome was change in leukocyte telomere length between baseline, year 2, and year 4. Intervention effects were analysed using mixed-effects linear regression models to account for repeated measurements within individuals over time.
A total of 2,571 telomere measurements were obtained from 1,031 participants across the three time points. Analyses compared telomere attrition trajectories among participants assigned to vitamin D₃ supplementation, omega-3 fatty acid supplementation, combined supplementation, or placebo.
Results
Over the four-year follow-up period, participants randomized to vitamin D₃ supplementation exhibited a statistically significant attenuation of leukocyte telomere length attrition compared with those assigned to placebo.
Vitamin D₃ supplementation was associated with a reduction in telomere attrition of approximately 0.14 kilobase pairs (140 base pairs) over four years relative to placebo (95% confidence interval: 0.007 to 0.27 kilobase pairs; p = 0.039). Trend analyses indicated that telomere length was approximately 0.035 kilobase pairs higher per year among participants receiving vitamin D₃ compared with placebo (95% confidence interval: 0.002 to 0.07 kilobase pairs; p = 0.037).
Marine omega-3 fatty acid supplementation did not demonstrate a statistically significant effect on leukocyte telomere length at either year 2 or year 4. Combined supplementation did not produce effects beyond those observed for vitamin D₃ alone within the study timeframe.
Measurement considerations and limitations
Leukocyte telomere length measured by qPCR reflects an aggregate signal influenced by both telomere dynamics and circulating immune cell composition. Changes in leukocyte subtypes may contribute to observed differences independently of telomere maintenance within specific cell populations.
While qPCR-based methods enable efficient longitudinal assessment in large cohorts, they are less precise than alternative techniques such as Southern blot terminal restriction fragment analysis or flow cytometry–based approaches. Findings are therefore interpreted at the group level rather than as exact telomere measurements in individuals.
As an ancillary study, the telomere cohort represents a subset of the parent VITAL population. Selection factors and representativeness should be considered when generalising results beyond the studied group.
Generalisability and population context
The study population consisted of older adults, with enrolment beginning at ages 50 to 55 years depending on sex. Telomere dynamics observed in this context reflect ageing-related biological processes within later adulthood.
Findings may not generalise to younger populations, to individuals with substantially different baseline vitamin D status, or to populations with different genetic, environmental, or lifestyle backgrounds.
Mechanistic inference boundary
This study did not test biological mechanisms linking vitamin D₃ exposure to telomere biology. The observed association describes differences in telomere attrition trajectories under randomized exposure conditions and remains consistent with, but does not confirm, hypotheses relating vitamin D signalling to cellular maintenance environments.
No mechanistic pathway can be inferred from these data.
Interpretation within vitamin D physiology
Vitamin D biology interacts with multiple regulatory systems that evolve with age, including immune cell function, inflammatory signalling, and endocrine feedback mechanisms. The observed association between vitamin D₃ exposure and telomere attrition patterns should be interpreted within the broader context of age-related changes in vitamin D handling and responsiveness.
For a physiological overview of how age modifies vitamin D biology, see Vitamin D and age.
Interpretation
Within the context of a large randomized controlled trial, vitamin D₃ exposure was associated with a modest attenuation of leukocyte telomere attrition over four years. These findings describe population-level biomarker dynamics under controlled exposure conditions and do not establish clinical benefit, ageing modification, or individual health effects.
The results should be interpreted strictly within the boundaries of biomarker research, study duration, population characteristics, and measurement methodology.
Primary source and publication details
Zhu H, Manson JE, Cook NR, et al.
Vitamin D₃ and marine ω-3 fatty acids supplementation and leukocyte telomere length: 4-year findings from the VITamin D and OmegA-3 TriaL (VITAL) randomized controlled trial.
American Journal of Clinical Nutrition. 2025.
DOI: 10.1016/j.ajcnut.2025.05.003