Silk's well-defined response to environmental conditions makes it a useful candidate to investigate the relationship between structure and function in biological materials. This area of research is of increasing importance as commonly employed microscopic and spectroscopic techniques often demand samples to be exposed to environments quite unlike those found in nature (i.e. low humidities and vacuum pressures). Whilst these conditions may enable high-quality structural data, their effects on a sample's mechanical properties are not yet fully understood. Using in situ tensile testing, we determine the effects of sample preparation and environment on individual fibres of Bombyx mori silk under conditions suitable for environmental scanning electron microscopy. We report significant differences in mechanical properties of the silk, depending on both the sample preparation (coating and fibre mounting) and environment (vacuum, imaging gas and pump-down procedure). We interpret these differences within the context of sample hydration by comparison with ex situ stress-strain analysis of B. mori silk under conditions ranging from 10 to 80 % relative humidity. We conclude that silks, with their ready availability and ease of preparation, are an ideal validation material for future technique developments in this area.