The effect of riders and handlers on horses has come under increasing scrutiny as the technology to measure physical horse human interaction has become more readily available. Reins are commonly used to deliver signals and instruction to horses through the application and release of tension and pressure. To date all rein tension studies, such as those examining the influence of physical attributes of reins and their use in different gaits, have relied on 2-handed rein use. Since effective rein use is crucial for the delivery of clear, distinguishable signals and subsequently discernible release of pressure when the required response is given by the horse, the aim of this study was to examine the effectiveness of rein use when held in 2 hands as most riding instruction dictates or when held in a single hand as required in some equestrian disciplines. Twenty female riders (28.2 ± 10.1 years), self-rated as novice or experienced, representing a wide range of equestrian disciplines sat in a general purpose saddle secured on stationary fixed saddle stand with the stirrups set at their usual riding length. Centaur Rein Tension gauges were fixed to a solid box at horse head height and fitted with a standard pair of leather 5 × 20 mm wide reins. Participants were asked to take up the rein tension that they would use when riding in trot. This was repeated 3 times per condition (using both hands and one-handed, using the riders natural hand of choice) using a cross-over design with a 30-minute wash-out period. Rein tension data (N) were analyzed using parametric paired t tests. No significant differences were evident between left and right hand rein tension when using 2 hands (t59 = 1.89; P > 0.05; left = 4.73 ± 2.65 N; right = 4.91 ± 2.63N) or 1 hand (t59 = 0.20; P > 0.05; left = 4.21 ± 2.32 N; right = 4.17 ± 2.91 N). Comparison of left:right hand difference in rein tension for 2-handed versus 1-handed test conditions was also nonsignificant (t38 = 0.63; P > 0.05). Although there was less absolute difference between left and right hand rein tension when riding with one hand than with 2 hands, the holding of the left and right reins was slightly less consistent with 1 hand than with 2, but not significantly so. Greater understanding of the direct effect of how riders hold the reins on clarity of signals relayed and pressure-release achieved is essential to safeguard horse welfare. Reliable rein tension studies that include different riding styles are required to improve understanding of the rider's effect on the horse in order to bring about changes in practice that will improve the welfare of horses used in equestrianism.