Background: Footwear interventions are often prescribed to assist with the management of lower limb pain, injury and disease. Commercially available shoe insoles and orthoses are increasingly incorporating novel design features to alleviate foot and lower limb symptoms, but this may be at a cost to optimal functional performance. This study compared the immediate effects of wearing glycerine-filled insoles, contoured prefabricated orthoses, and flat insoles, on balance and gait measures. Methods: Thirty healthy adults (17 men, 13 women; mean [SD] age: 24.3 [2.5] years) performed tests of single-leg standing with eyes open (Kistler force platform), star excursion balance test, and level-ground walking (GAITRite® walkway system), under three randomised conditions: wearing glycerine-filled insoles, prefabricated orthoses, and flat (control) insoles, within their own footwear. Centre of pressure movement (anterior-posterior and mediolateral range and standard deviation, total path velocity), star excursion balance test reach distance, and temporospatial gait variables were collected. Perceived comfort of the inserts was scored immediately after use on a 100 mm visual analogue rating scale. After trialling all inserts each participant ranked their level of comfort from least to most. Results: Centre of pressure measures, star excursion balance test reach distance, or temporospatial gait variables did not differ between the three inserts (all P values >0.088). Significant between-condition differences were reported for comfort ranking (P = 0.031), but not rating scores (P = 0.638). Weak to moderate negative correlations (r values ranged between -0.368 and -0.406) were observed between visual analogue scale comfort rating for the flat insoles and prefabricated orthoses, star excursion balance test and gait measures. Conclusions: Single-leg standing balance, star excursion balance test performance, and level-ground walking patterns in asymptomatic adults do not appear to differ when wearing glycerine-filled insoles, contoured prefabricated orthoses, or flat insoles. Perceived comfort may be related to the biomechanical or clinical effectiveness of novel footwear interventions, and requires further investigation. Importantly, these findings are specific to a healthy population and further research is needed to determine the long-term effects of glycerine-filled insoles in patients with known balance impairments.