Tract tracing with neuronal tracers not only represents a straightforward approach to identify axonal projection connection between regions of the nervous system at distance but also provides compelling evidence for axonal regeneration. An ideal neuronal tracer meets certain criteria including high labeling efficacy, minimal neurotoxicity, rapid labeling, suitable stability in vivo, and compatibility to tissue processing for histological/immunohistochemical staining. Although labeling efficacy of commonly used fluorescent tracers has been studied extensively, neurotoxicity and their effect on neural functions remains poorly understood. In the present study, we comprehensively evaluated motor and sensory nerve function 2-24 weeks after injection of retrograde tracer Fluoro-Gold (FG), True Blue (TB) or Fluoro-Ruby (FR) in the tibial nerve in adult Spague-Dawley rats. We found that motor and sensory nerve functions were completely recovered by 24 weeks after tracer exposure, and that FG lead to a more prolonged delay in functional recovery than TB. These findings shed light on the long-term effect of tracers on nerve function and peripheral axonal regeneration, and therefore have implications in selection of appropriate tracers in relevant studies.