Aim <p>To investigate the impact of prolonged running on speed at the moderate-to-heavy intensity transition, and whether increased eccentric load modifies exercise-induced shifts in the moderate-to-heavy intensity transition.</p> Methods <p>Twelve endurance-trained runners (4 females, 8 males, peak oxygen uptake 51.5&#xa0;mL kg⁻¹ min⁻¹ in females and 56.1&#xa0;mL kg⁻¹ min⁻¹ in males) participated. Following trials to assess speed at the first ventilatory threshold (VT₁) on a 0% gradient and -10% gradient, participants completed two trials: (i) level trial (LVL): 120-min of running at 0% gradient and (ii) downhill trial (DH): 120-min of running with 90-min at 0% gradient and 6 × 5-min intervals at -10% gradient (DH). Running was conducted at 90% of VT₁ speed for respective gradients. Speed at VT₁ on 0% gradient, perceived leg muscle soreness, and maximal voluntary isometric knee extensor torque were assessed pre- and post-prolonged exercise.</p> Results <p>An effect of time was observed for speed at VT₁ (∆-6.2 ± 3.6% in LVL and ∆-7.6 ± 3.2% in DH, <i>P</i> &lt; 0.001), with no effect of condition (LVL vs. DH,<i> P</i> = 0.382), or time × condition interaction (<i>P</i> = 0.295). Reduced metabolic energy expenditure (metabolic power) significantly contributed to the reduced speed at VT₁ in both trials (<i>P</i> &lt; 0.001), whereas changes in running economy (energetic efficiency) did not (<i>P</i> = 0.228).</p> Conclusion <p>Prolonged moderate-intensity running reduced speed at the moderate-to-heavy intensity transition, but this was not exacerbated by increased eccentric load. Reduced speed at the moderate-to-heavy intensity transition following prolonged running was primarily attributed to decreased metabolic power.</p>