Catalyst deactivation is a major problem in Fischer-Tropsch synthesis. It leads to a decrease in hydrocarbon productivity and loss of active sites in the expensive cobalt catalyst, and thus, undermines the overall efficiency of the technology. In the present paper, the effect of deactivation of silica-supported cobalt catalysts and their reductive rejuvenation on the number of active sites and their intrinsic activity (turnover frequency) in Fischer-Tropsch synthesis was studied using a combination of Steady State Isotopic Transient Kinetic Analysis (SSITKA) and catalyst characterization techniques. Catalyst characterization revealed that carbon deposition and agglomeration of cobalt nanoparticles during reaction were responsible for the deactivation. SSITKA experiments showed that the initial rate constant of 2.33 μmol g−1 s−1 had a loss of 67% of activity after 150 h on stream with a reduction in the amount of sites due to deposited carbon by 33.4 μmol g−1. The carbon deposition leads to a decrease in the number of carbon chemisorbed intermediates which yield methane through their hydrogenation and desorption. The number of sites for reversible adsorption of CO is less affected by carbon deposition. The surface hydrogenation sites and surface sites favoring stronger reversible adsorption of carbon monoxide deactivate first during the first hours of Fischer-Tropsch synthesis. Catalyst rejuvenation in hydrogen lessens the amounts of deposited carbon species and partially releases the most active sites of carbon monoxide dissociative adsorption and stronger sites of carbon monoxide reversible adsorption. The transient isotopic methods provide an attractive tool to obtain precise information about the mechanisms of deactivation of cobalt catalysts in Fischer-Tropsch synthesis.