Published Sep 06, 2026 | 7:30 AM ⚊ Updated Sep 06, 2026 | 7:30 AM
D3 rehabilitation restored muscle fibre area, while D2 alone did not produce a measurable restoration.
Synopsis: A Hyderabad study found that while vitamin D2 and D3 worked similarly on calcium-related measures in male rats, D3 was more effective across several muscle and heart measures, especially during recovery from vitamin D deficiency. Researchers caution that the findings need human studies before influencing supplementation or fortification recommendations.
Vitamin D is usually discussed in the language of bones, calcium and deficiency. But a new study from Hyderabad suggests that the form of vitamin D may matter when the focus shifts to what happens inside muscles and the heart.
Researchers at the ICMR-National Institute of Nutrition (ICMR-NIN), Hyderabad, found that vitamin D3 was more effective than vitamin D2 across several measures of skeletal muscle and cardiac structure, function and metabolism in male rats. The difference was particularly clear when the animals were recovering from vitamin D deficiency.
The study, published in Molecular Nutrition & Food Research, compared vitamin D2, vitamin D3 and a combination of both forms in rats. The researchers found that the two forms worked similarly on calcium regulation, but D3 had a stronger effect on several measures beyond calcium.
Dr Ayesha Ismail, Scientist at ICMR-NIN and the study’s corresponding author, said the distinction was important because the two forms did not behave identically outside their traditional role in calcium regulation.
“Although both forms of vitamin D were effective in improving calcium-related parameters, vitamin D2 was less effective than vitamin D3 in several parameters related to muscle and heart structure and energy metabolism,” she said.
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The study used weaning male rats and tested what happened when they received different forms of vitamin D over time.
Some rats received vitamin D2, some vitamin D3, some received both, while another group was kept on a vitamin D-deficient diet. The researchers then used a second phase to test whether changes caused by deficiency could be reversed.
Rats that developed vitamin D deficiency were placed on rehabilitation diets containing D2, D3 or both forms for another nine weeks. The researchers assessed body composition, grip strength, blood markers, muscle tissue and the left ventricle of the heart.
The idea was straightforward: if vitamin D2 and D3 are doing the same job, would they also be equally good at helping tissues recover after deficiency?
The answer was not always yes.
One of the first differences appeared in the blood.
The researchers found that the 25-hydroxylated form of vitamin D3 was significantly higher in serum than the corresponding form produced from vitamin D2. This means that the same dietary comparison did not result in identical circulating vitamin D metabolites.
The researchers point to differences in how the two forms are processed and transported in the body. They note that CYP2R1, a major liver enzyme involved in vitamin D metabolism, and vitamin D-binding protein have greater affinity for D3 than D2.
But the researchers did not directly test whether this difference was responsible for D3’s stronger effects.
The important point was that this difference in vitamin D levels did not translate into a major separation when it came to calcium regulation.
“Both forms were effective in improving calcium-related parameters,” Ismail said.
That changed when the researchers looked at muscle.
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Vitamin D deficiency affected the rats beyond their calcium levels.
The deficient animals had lower lean body mass, smaller muscle fibres and weaker grip strength. When vitamin D was restored, D3 produced stronger improvements across several of these measures.
The effect was particularly visible in the group receiving 2,000 IU/kg of vitamin D3 during rehabilitation. These rats regained total body mass and lean mass to levels closer to animals that had received D3 from weaning. The D2 groups showed smaller gains.
Muscle fibre size offered another clear difference.
On tissue examination, D3 rehabilitation restored muscle fibre area, while D2 alone did not produce a measurable restoration to the same extent.
Grip strength also improved during rehabilitation with both forms, but the strongest improvement was seen with the higher-dose D3 group.
The researchers found similar differences in some molecular and metabolic measures of muscle.
The study reported that vitamin D2 was less effective than D3 at improving muscle fibre size, strength, expression of contractility-related genes and metabolic enzymes.
Citrate synthase, an enzyme involved in cellular energy production, was another example. Its activity increased with D3, while D2 alone produced much smaller changes during rehabilitation.
Not every marker behaved identically, however. The researchers found differences across specific genes and enzymes rather than one uniform response across every measure.
That distinction matters because the study was not simply showing that D3 “works” and D2 “doesn’t”. Both forms had biological effects. D3 was stronger across several measures.
The researchers then looked at the heart, focusing on molecular markers linked to cardiac contraction and metabolism.
Vitamin D deficiency altered several of these markers, including Myh7, Tnnt2, Ttn and Serca2a. During recovery, the higher-dose D3 group showed stronger improvements in several of these measures than the corresponding D2 group.
One marker, Serca2a, is involved in handling calcium inside heart muscle cells and is important for contraction and relaxation.
Myh7 expression was also higher in rats receiving 2,000 IU/kg of D3 than in those receiving the same dose of D2 during recovery.
Dr Bharati Kulkarni, Director of ICMR-NIN, said the findings suggest that the difference between the two forms may extend beyond the traditional role of vitamin D in bones and calcium.
“The findings indicate that vitamin D3 may be more effective than vitamin D2 in maintaining and restoring skeletal muscle and cardiac functions,” she said.
However, the findings should not be interpreted as evidence that D3 prevents heart attacks or heart failure.
The researchers measured molecular and tissue-level indicators of cardiac function and metabolism. They did not conduct clinical cardiovascular outcome testing.
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The heart findings extended to tissue changes associated with fibrosis.
Vitamin D-deficient rats showed increased collagen deposition around blood vessels in the left ventricle. When the deficient animals were placed on rehabilitation diets, tissue staining showed that both D3 diets reduced the fibrotic changes seen around blood vessels in the heart, while D2 did not.
D2 did not produce the same tissue-level reversal.
But another measurement complicated the picture.
Hydroxyproline, a chemical marker associated with collagen in tissue, remained elevated in the deficient range during rehabilitation. Neither D2 nor D3 normalised this measure.
So while D3 improved the appearance of perivascular fibrosis on tissue staining, the study did not show that every indicator of cardiac fibrosis returned to normal.
D2 and D3 are closely related forms of vitamin D, but they are not chemically identical.
The researchers point to structural differences between the molecules that can affect how they are processed in the body.
Their paper notes that the liver enzyme CYP2R1 and vitamin D-binding protein have greater affinity for D3 than D2. The researchers suggest that this could contribute to the higher circulating levels of the vitamin D form produced in the blood after D3 is processed seen with D3.
That could, in turn, help explain why D3 showed stronger effects in some muscle and heart measures.
But this is still a proposed explanation, not a proven mechanism.
This is where the study’s limits become important.
The experiment was conducted entirely in male rats. It does not establish that vitamin D3 will produce the same advantages over vitamin D2 in humans.
The researchers also did not test women or determine whether the same tissue responses would occur in females.
Other limitations included the inability to measure 1,25-dihydroxyvitamin D, the active hormonal form of vitamin D, and the absence of electrocardiography to directly assess heart rhythm.
The study therefore does not provide a basis for saying that people should switch from D2 to D3 specifically to protect their muscles or heart.
Instead, it adds to the evidence that the two forms of vitamin D may not be biologically interchangeable in every tissue or situation.
The researchers say more work is needed before the findings can inform supplementation or food-fortification policies.
“Further studies are needed to establish the effects of the two forms of vitamin D on skeletal and extra-skeletal functions and their implications for supplementation and fortification programmes,” Dr Kulkarni said.
For now, the Hyderabad study draws a distinction between two things that are easy to lump together: correcting vitamin D deficiency and understanding what different forms of the vitamin do inside the body.
In these male rats, D2 and D3 were similarly effective at regulating calcium-related measures. But when researchers looked beyond calcium, D3 showed stronger effects across several measures of skeletal muscle and cardiac structure, function and metabolism, particularly during recovery from deficiency.
Whether that same advantage exists in humans remains an open question. The researchers say further studies will be needed before the findings can influence supplementation or fortification recommendations.
