Objectives To examine the level of loneliness experienced during the COVID-19 pandemic in Denmark and to identify associated behavioural patterns and demographic factors. Design Cross-sectional cohort study. Setting Includes Danish active and former blood donors. Participants A questionnaire was sent to 124 307 active and former blood donors, of these a total of 50 968 participants completed the study questionnaire (response rate=41%). Primary and secondary outcome measures Subjective experience of loneliness was measured using the 3-item University of California, Los Angeles Loneliness Scale (UCLA-3). Besides the UCLA-3, the respondents answered items on sociodemographic and economic characteristics, items on precautionary measures taken to avoid COVID-19 infection as well as on COVID-19 anxiety. Results The participants indicated their experienced level of loneliness both before and during the pandemic. Comparing the two reports yielded a mean increase in loneliness scores of 14.1% (p<0.001). Exploratory factor analysis identified the factor well-being, which comprised three questionnaire items related to emotional heath, physical health and happiness. A high score on the factor well-being was associated with reduced levels of loneliness (coefficient=-0.47, 95% CI -0.49 to -0.46)). Furthermore, women were more likely than men to have experienced increased levels of loneliness during the pandemic (coefficient=0.27, 95% CI 0.25 to 0.29). Furthermore, a negative correlation between higher age and change in loneliness score was observed. Conclusions The findings document an increase in the level of experienced loneliness during the COVID-19 pandemic, particularly affecting individuals with low well-being, women and younger individuals.