Mobile Laser Scanning (MLS) is rapidly increasing in popularity for the capture and quantification of vegetation structure in natural landscapes. However, understanding the optimal implementation for forest capture in the field is still limited, particularly regarding the choice in acquisition path and length followed during data capture. How variable is the characterisation of vegetation structure when using different acquisition paths, and is this difference likely to be significant for most users? In this study we compared four acquisition path designs commonly cited in the literature to determine the importance of this choice for users when capturing vegetation structure. MLS point clouds were systematically captured to repeatedly survey study plots in a closed-canopy forest ecosystem in south-eastern Australia. Digital elevation models, canopy height models, and vertical voxel occupancy profiles were derived to illustrate the sensitivity of the path's length and configuration to variability in the quantification of vegetation structure. We found strong agreement between the acquisition path designs that increased as path length increased. No significant differences were found in digital elevation models derived at 75% and 100% path lengths. However, differences were observed in two and four of the 25% and 50% path length plots respectively. Significant differences in canopy height model data were only found in one plot at the 50% path length. Mean differences to the reference digital elevation models was 0.09 m across all plots, and 0.4 m for the canopy height models. Voxel occupancy profiles showed the greatest agreement between the understory and canopy where the mean difference between acquisition paths across all plots was 5.72%. Our findings have significant implications for the use of MLS from an operational perspective, as they illustrate the variability and reliability of their use in natural forested landscapes. Users can choose to balance their choice in acquisition path design with path length to meet requirements such as efficiency or ease of data capture in the field.