BACKGROUND: This is the first study analyzing levels of physical activity in a sample of quarantined adults with chronic conditions. The aim of this study was to compare moderate- and vigorous-intensity physical activity levels in Spanish adults with chronic conditions before and during coronavirus disease 2019 (COVID-19) quarantine. METHODS: A cross-sectional online survey was administered during the COVID-19 quarantine in Spain. A total of 163 participants with chronic conditions (113 females and 47 males; age range 18-64 years) completed the survey. A total of 26 chronic conditions were included. Participants self-reported average minutes/day of moderate and vigorous physical activity before and during quarantine. Differences in moderate- and vigorous-intensity physical activity levels before and during COVID-19 quarantine (overall, by gender, by age, by number of chronic conditions and by each chronic condition) were assessed by Wilcoxon-signed rank test. RESULTS: During COVID-19 quarantine, there was a significant decrease of moderate-intensity physical activity in Spanish people with chronic conditions (in both males and females, in those aged 18-24, 25-34, 35-44 and 55-64 years, in those with multimorbidity, in those with one/two chronic condition/s, and in those diagnosed with asthma/hypercholesterolaemia/chronic skin disease/haemorrhoids). Also, there was a significant decrease of vigorous-intensity physical activity in Spanish males with chronic conditions and in those with multimorbidity. CONCLUSIONS: These results should be considered to develop effective strategies of physical activity promotion targeting these specific groups when new quarantine or restriction measures are implemented, in order to avoid new significant decreases of physical activity in these vulnerable populations.