Generally
Use a variable that holds the last records' value to calculate the delta
select some_column,
some_column - @prev as delta,
@prev := some_column
from your_table, (select @prev := 0) p
SQLFiddle demo
The actual query
SELECT DAY(`timestamp`) as day_ts,
floor(HOUR(`timestamp`)/2) as hour_ts
sum(total_users)
sum(total_users) - @prev AS col1,
@prev := sum(total_users)
FROM stats, (select @prev := 0) p
GROUP BY day_ts, hour_ts