The aim of this study was to evaluate the feasibility of methods for objective 24-h sampling of physical activity among cleaners. Twenty cleaners participated in three 24-h measurements. Amount of steps, heart rate (HR), cardio-respiratory fitness, body mass index (BMI) and blood pressure were measured. The methods were feasible for the objective 24-h sampling of physical activity and cardio-respiratory fitness among cleaners. Measurements showed that the cleaners walked 20,198 ± 4,627 steps per day. During working hours, the average cardio-respiratory load was 25 ± 6% of heart rate reserve (HRR). The cleaners had a low estimated cardio-respiratory fitness (34 mlO2/kg/min), a high BMI (50%, >25 kg/m(2)) and blood pressure (50%, >120/>80 mmHg). The high amount of steps, the relatively high cardiovascular load at work and low cardio-respiratory fitness illustrate the need for further investigation of the relationship between physical activity at work and in leisure, and cardiovascular health in this population. Practitioner Summary: This study evaluated the feasibility of methods for objective 24-h sampling of physical activity among cleaners; the methods used were found to be feasible. The cleaners had a high cardiovascular load at work and low cardio-respiratory fitness, suggesting that there is a need for further investigations.