By performing unsteady CFD simulations using RNG k–ɛ model and dynamic mesh technique, this paper investigates how the walking motion of health care worker (HCW) influences gaseous dispersion in a six-bed isolation room with nine downward supplies and six ceiling-level or floor-level exhausts. The flow near and behind HCW is easily affected by HCW motion. The flow disturbance induced by HCW walking with swinging arms and legs is a mixing process. The walking HCW displaces air in front of it and carries air in the wake forwardly, meanwhile pressure difference drives air from two lateral sides into the wake. HCW motion (0–5.4 s) indeed induces a little gaseous dispersion, but the residual flow disturbance after HCW stops (5.4 s–25.4 s) induces more gaseous agent spread and it requires more than 30–60 s to approximately recover to the initial state after HCW stops. Although HCW motion indeed affects airborne transmission, but its effect is less important than ventilation design. No matter with or without HCW motion, the ceiling-level exhausts perform much better in controlling airborne transmission than the floor-level exhausts with the same air change rate (12.9 ACH). Smaller air change rate of 6 ACH experiences higher concentration and more gaseous spread than 12.9 ACH. In contrast to the realistic human walking, the simplified motion of a rectangular block produces stronger flow disturbance. Finally surface heating of HCW produces a stronger thermal body plume and enhances turbulence near HCW, thus slightly strengthens airborne transmission. •Human motion indeed affects airflows and airborne spread in a six-bed isolation room.•But ventilation design is more significant than human motion for gaseous spread.•The flow near and behind HCW is easily affected by HCW motion with a mixing process.•The residual flow after HCW motion induces more gaseous spread than during it.•The simplified motion of a rectangular block produces stronger flow disturbance.