(1) Background: The vitamin D level in blood is one of the markers of the functional reserves of the human body and can contribute to more successful adaptation in the Arctic. (2) Methods: The study involved 38 participants in the project “Arctic Floating University—2021”. The determination of vitamin D content was carried out at the beginning of the expedition. A dynamic study was carried out for 20 days in the morning and in the evening. The functional state parameters of the participants were assessed using psychophysiological and questionnaire methods. Statistical methods: Mann–Whitney U-test and correlation analysis. (3) Results: It was found that at the beginning of the expedition, the functional state of participants with more severe vitamin D deficiency is characterized by a shorter average duration of RR intervals (p= 0.050) and reduced SDNN values (p= 0.015). The higher the content of vitamin D, the greater increase in speed (r= 0.510), the higher the increase in projective performance (r= 0.485), and the smaller the increase in projective stress (r= −0.334). Significant relationships between the subjective characteristics of functional states and the vitamin D of participants have not been established. (4) Conclusion: With an increase in the severity of vitamin D deficiency in the blood, the adaptive capabilities of participants decrease during an expedition to the Arctic.