Sweat testing is often conducted to assist with electrolyte replacement plans for athletes. However, the effect of patch application timing and on-skin duration on local sweating rate (LSR) and sweat electrolyte concentrations is unclear. Purpose: To determine the effect of patch application timing and on-skin duration on LSR and local sweat [Na⁺], [K⁺], and [Cl^-]. Methods: Thirty-nine recreationally trained (VO₂max: 47.1±7.8 ml/kg/min) athletes (27 M, 12 F; 75.4±12.4 kg) cycled at ~80% HR_max in the heat (32°C, 39% rh). Prior to (PRE) and 15 min into exercise (EX), two sweat patches were applied to the left and right mid-back, respectively. The patches were removed after a skin adherence duration of 30 (SHORT) or 70 (LONG) min. LSR was equated from sweat mass over patch surface area (11.9 cm²) and duration. Sweat was centrifuged and analyzed for [Na⁺], [K⁺], and [Cl^-] by ion chromatography. A two-way repeated measures ANOVA was used to determine the effect of patch application timing (PRE vs. EX), duration (SHORT vs. LONG), and interaction effects on each variable, followed by Tukey post-hoc where main effects were found. Significance was set at p<0.05. Results: There was an interaction effect with EX LONG and EX SHORT > PRE LONG > PRE SHORT for [Na⁺] (56.8±21.6, 58.5±22.3 > 50.7±20.1 > 46.8±19.6 mmol/L, p<0.0001), [Cl^-] (55.2±23.5, 53.5±25.1 > 49.4±22.1 > 38.2±21.7 mmol/L, p<0.0001), and LSR (1.4±0.3, 1.6±0.6 > 1.1±0.3 > 0.8±0.4 mg/cm²/min, p<0.0001). There were no significant differences for [K⁺] between EX LONG, EX SHORT, PRE LONG, AND PRE SHORT (3.8±0.6, 4.0±0.9, 3.6±0.6, 3.3±0.6 mmol/L, p=0.79). Conclusion: The on-skin duration did not affect sweat [Na⁺] and [Cl^-] when patches were applied during exercise. However, applying patches prior to exercise resulted in lower sweat [Na⁺] and [Cl^-], especially when removed after a short duration. This was likely due to lower LSR during the ramp up to steady state sweating. Therefore, practitioners should take patch application timing into account when interpreting sweat electrolyte results. Local sweat [Na⁺] and [Cl^-] measured from patches applied prior to exercise may not be representative of concentrations during the full bout of exercise. However, more research is needed to determine the impact of patch timing in the context of whole body sweat [Na⁺] and [Cl^-] estimations.