Trekking and military missions generally consist of carrying heavy loads for extreme durations. These factors have been separately shown to be sources of neuromuscular (NM) fatigue and locomotor alterations. However, the question of their combined effects remains unresolved, and addressing this issue required a representative context. PURPOSE: The aim was to investigate the effects of extreme-duration heavy load carriage on NM function and walking characteristics. METHODS: Ten experienced infantrymen performed a 21-h simulated military mission (SMM) in a middle-mountain environment with equipment weighing ∼27 kg during battles and ∼43 kg during marches. NM function was evaluated for knee extensors (KE) and plantar flexors (PF) pre- and immediately post-SMM using isometric maximal voluntary contraction (MVC) measurement, neural electrical stimulation and surface EMG. The twitch-interpolation method was used to assess central fatigue. Peripheral changes were examined by stimulating the muscle in the relaxed state. The energy cost, mechanical work and spatio-temporal pattern of walking were also evaluated pre-/post-SMM on an instrumented treadmill in three equipment conditions: Sportswear, Battle and March. RESULTS: After the SMM, MVC declined by -10.2±3.6% for KE (P\textless0.01) and -10.7±16.1% for PF (P = 0.06). The origin of fatigue was essentially peripheral for both muscle groups. A trend toward low-frequency fatigue was detected for KE (5.5%, P = 0.08). These moderate NM alterations were concomitant with a large increase in perceived fatigue from pre- (rating of 8.3±2.2) to post-SMM (15.9±2.1, P\textless0.01). The SMM-related fatigue did not alter walking energetics or mechanics, and the different equipment carried on the treadmill did not interact with this fatigue either. CONCLUSION: this study reports the first data on physiological and biomechanical consequences of extreme-duration heavy load carriage. Unexpectedly, NM function alterations due to the 21-h SMM were moderate and did not alter walking characteristics. CLINICAL TRIAL REGISTRATION: Name: Effect of prolonged military exercises with high load carriage on neuromuscular fatigue and physiological/biomechanical responses. Number: NCT01127191.