The purpose of this study was to evaluate whether treatment time and concentration of these reagents have an effect on the resulting gliding resistance. Forty peroneus longus (PL) tendons were used, from 20 adult mongrel dogs, along with the A2 pulley obtained from the ipsilateral hind paw. After the baseline gliding resistance was measured, the PL tendons were treated with one of three concentrations of hyaluronic acid (HA) and 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide (NHS) mixed with 10% gelatin for various times (5, 30, and 60 min). Tendon friction was measured over 1000 cycles of simulated flexion/extension motion. Gliding resistance of the untreated PL tendons had no significant difference among the groups. After surface treatment with low concentration of HA and EDC/NHS for 5-min cure, the gliding resistance was similar to that of the untreated PL tendon and significantly higher than its 30- and 60-min treatment. For the rest of high concentration of HA and EDC/NHS groups, the gliding resistance was lower than that of untreated PL tendon. However, there was no significant difference among the timing points. It is possible to optimize the effect of surface treatment on friction and durability by regulating cure time and concentration of reagents in a canine extrasynovial tendon in vitro.