The purpose was to examine and compare the effects of replacing time spent sitting with standing at work on fat-free mass, fat mass and waist circumference using isotemporal substitution. Analyses were conducted on work hours on both cross-sectional and longitudinal data. The study included 223 persons from an intervention study aimed at reducing sitting time at work among office employees. Sitting, standing and anthropometry were measured objectively. Cross-sectional isotemporal substitution analyses were modelled on baseline data, while longitudinal analyses were modelled based on differences in sitting and standing time at work between baseline and 1-month follow-up in relation to differences in anthropometric measures between baseline and 3-months follow-up. Replacing one hour of sitting time with one hour of standing was associated with a 0.21 kg higher fat-free mass in the longitudinal analysis and 0.95 kg in the cross-sectional analysis. Fat mass was 0.32 kg lower in the longitudinal analysis and 0.61 kg lower in the cross-sectional analysis. Waist circumference decreased by 0.38 cm in the longitudinal analysis and 0.81 cm in the cross-sectional analysis. Both cross-sectional and longitudinal analyses showed an effect on body composition measures by replacing one hour of sitting with standing however, this effect was largest in the cross-sectional analyses. Trial registration ClinicalTrials.gov NCT01996176.