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This study examined the effect of repeated bouts of level and downhill running on physiological markers of effort and exercise-induced muscle soreness in trained female distance runners. Ten participants (Age: 24.4 ± 2.0 years; V̇O_2peak: 52.9 ± 1.1 mL·kg⁻¹·min⁻¹), naïve to downhill running, completed six alternate 5 min trials of level and downhill running (−15%) at a 70% velocity at V̇O_2peak on two occasions, three weeks apart. Perceived muscle soreness was measured upon completion and in the 72 h post exercise. V̇O₂, Heart Rate (HR), Blood Lactate (BLa), and Respiratory Exchange Ratio (RER) were lower running downhill (<i>p</i> < 0.016, ηp² > 0.541). For the first downhill run, Rating of Perceived Exertion (RPE) was higher compared to that for level running (<i>p</i> = 0.051; <i>d =</i> 0.447), but for the remaining trials, RPE was lower when running downhill (<i>p</i> < 0.004; <i>d ></i> 0.745). V̇O₂, HR, and RER were not different in the second bout (<i>p</i> > 0.070, ηp² < 0.318); however, V̇O₂ was lower in each downhill trial (Δ = 1.6–2.2 mL·kg⁻¹·min⁻¹; <i>d</i> = 0.382–0.426). In the second bout, BLa was lower (<i>p</i> = 0.005, ηp² = 0.602), RPE in the first trial was lower (<i>p</i> = 0.002; <i>d =</i> 0.923), and post exercise perceived soreness of the gastrocnemius, quadriceps, and hamstrings was attenuated (<i>p</i> < 0.002; ηp² > 0.693). Perceived soreness of the gluteal muscles was lower in the second bout immediately post exercise, 24 h, and 48 h post exercise (<i>p</i> < 0.025; <i>d ></i> 0.922). A repeated bout of downhill running attenuated perceived muscle soreness and may modulate the physiological and perceived physical demand of a second bout of level and downhill running.