Background: Recent research indicates prolonged fasting for more than 7 days is beneficial in priming the vitamin D metabolism. However, methodological limitations in previous studies, such as insufficient sample size and a lack of control group, limits its generalizability. The present study evaluated the impact of prolonged fasting (10 days) on vitamin D, vitamin B12, body mass index (BMI), weight, hemoglobin, vitality and quality of life (QoL) compared to a normal diet. Methods: This randomized control trial was conducted in an in-patient private yoga and naturopathy setting between February and April 2022. A total of 52 participants (mean age 51.57 ± 13.67 years) with varying medical conditions were randomized into a fasting group (FG) or a normal diet group (NDG) with 26 participants in each group. The FG was on a fasting diet (500 kCal/day) which included holy basil herbal tea, lemon honey water, and water (3 L). The NDG (1500 kCal/day) consumed a routine diet that included Indian bread, pulses, steamed rice, vegetable salads, and beverages. Results: The FG has shown significant increase in the Vitamin D levels ( P = .003, d = 0.475), vitality ( P = .006, d = 0.425), physical QoL ( P < .001, d = 0.549), psychological QoL ( P = .002, d = 0.488), and environmental QoL ( P = .004, d = 0.457) compared to NDG. No significant changes were observed in vitamin B12, weight, BMI, haemoglobin, and social QoL. A weak to moderate (ρ = 0.330-0.483) positive correlation was observed between vitality scores and QoL domains, whereas BMI scores showed an inverse correlation (ρ = −.280) with vitamin D levels. Conclusion: The results suggest that prolonged fasting for 10 days can improve vitamin D levels, improve vitality, and promote quality of life compared to a normal diet. Unlike previous studies, the FG does not differ from the NDG concerning weight and BMI. Nevertheless, fasting may be utilized as an effective tool to tackle vitamin D deficiency and associated health insufficiencies.