The phase III slope of sulfur hexafluoride (SF₆) in a single breath washout (SBW) is greater than that of helium (He) under normal gravity (i.e. 1G), thus resulting in a positive SF₆-He slope difference. In microgravity (µG), SF₆-He slope difference is smaller due to a greater fall in the phase III slope of SF₆ than He. We sought to determine whether increasing thoracic fluid volume using 60-degree head-down tilt (HDT) in 1G would produce a similar effect to µG on phase III slopes of SF₆ and He. Single-breath vital capacity (SBW) and multiple-breath washout (MBW) tests were performed prior to, during, and 60 minutes after one-hour of HDT. Compared to baseline (SF₆ 1.050±0.182 %/L, He 0.670±0.172 %/L), the SBW phase III slopes for both SF₆ and He tended to decrease during HDT, reaching nadir at 30 minutes (SF₆ 0.609±0.211 %/L, He 0.248±0.138 %/L, p=.08 and p=.06, respectively). In contrast to µG the magnitude of the phase III slope decrease was similar for both SF₆ and He, therefore no change in SF₆-He slope difference was observed. MBW analysis revealed a decrease in normalized phase III slopes (Sn_III) at all time points during HDT, for both SF₆ (p<0.01) and He (p<0.01). This decrease was due to changes in the acinar, and not the conductive, component of Sn_III. These findings support the notion that changes in thoracic fluid volume alter ventilation distribution in the lung periphery, but also that demonstrate the effect during HDT does not wholly mimic that observed in µG.