Neuromuscular activation can be influenced by both muscle temperature (Tm) and fatigue. To assess the effects of Tm and fatigue on the electromechanical delay (EMD), which is commonly defined as the latency between the onset of muscle electrical activation and the onset of force output, 15 participants performed voluntary isometric contractions of different intensities under neutral (TmN), low (TmL) and high (TmH) Tm, before and after a fatiguing exercise. During contraction, electromyogram (EMG), mechanomyogram (MMG) and force (F) were recorded from the biceps brachii muscle. The overall EMD was calculated as the time lag between the onset of EMG and force signals and partitioned in the latencies between the onset of EMG and MMG (Dt EMG-MMG, which includes the electrochemical processes of EMD) and between the onset of MMG and F (Dt MMG-F, which includes the mechanical processes of EMD). Fatigue lengthened significantly to a similar extent EMD, Dt EMG-MMG and Dt MMG-F under all the investigated Tm. TmL elongated significantly Dt EMG-MMG, both before and after fatigue, but not EMD and Dt MMG-F. While fatigue affected EMD and all its components, muscle cooling altered the electrochemical (Dt EMG-MMG) but not the mechanical (Dt MMG-F) processes of EMD, suggesting that fatigue and cooling show similar effects on sarcolemmal propagation properties and excitation contraction process, but not on series elastic components characteristics.