Background: In the earlier developed and evaluated 12-week UPcomplish intervention, the aim was to reduce sedentary behaviour (SB) among office workers and increase their quality of life (QoL). In the current study, we explored moderators of effectiveness. Method: We applied a stepped wedge design with five intervention groups starting with time lags of seven weeks (n = 142, 96 females). Participants wore the VitaBit to continuously measure SB and received surveys about QoL and psychosocial determinants at the beginning, middle, and end of the intervention. We regressed baseline participant characteristics and behaviours onto intra-individual improvements (centred around calendar week means) in determinants, SB, performance objectives, and QoL. Results: Those scoring high in baseline intention, task performance, stress, vitality, and emotional well-being improved less in these variables. Baseline stress (β = − 0.05 [SE = 0.01; 95% CI = − 0.08, − 0.02; pcorrected =.02]) and emotional well-being (β = 0.02 [SE = 0.01; 95% CI = 0.01, 0.03; pcorrected =.02]) were associated with improvement in contextual performance. Baseline attitude (β = − 12.92 [SE = 3.93; 95% CI = − 20.80, − 5.04; pcorrected =.02]) and perceived behavioural control (PBC; β = − 9.27 [SE = 3.04; 95% CI = − 15.37, − 3.16; pcorrected =.03]) were negatively associated with improvements in emotional well-being. Post hoc analyses with a sub-group scoring lower in determinants revealed that improvement in PBC was positively associated with SB registration. Conclusion: Participants scoring low in baseline determinants might profit from UPcomplish via an increase in PBC. In combination with changes within organizations (e.g. the implementation of standing desks), UPcomplish might potentially reduce SB. Trial Registration: NL7503 — registered 1 February 2019. Highlights: Among less motivated participants, improving self-efficacy predicts improvement in sitting. Sedentary behavior might be more automatic and habitual and less intentional. Sitting less probably needs to be facilitated with environmental and cultural changes.